End-to-end cell therapy automation

ABSTRACT

The present disclosure provides an automated method of producing genetically modified immune cells, including chimeric antigen receptor T (CAR T) cells, utilizing a fully-enclosed cell engineering system.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.16/119,618, filed Aug. 31, 2018, which claims the benefit of U.S.Provisional Patent Application No. 62/670,391, filed May 11, 2018, andU.S. Provisional Patent Application No. 62/553,214, filed Sep. 1, 2017,the disclosures of each of which are incorporated by reference herein intheir entireties.

FIELD OF THE INVENTION

The present disclosure provides an automated method of producinggenetically modified immune cells, including chimeric antigen receptor T(CAR T) cells, utilizing a fully-enclosed cell engineering system.

BACKGROUND OF THE INVENTION

As anticipation builds about accelerated clinical adoption of advancedcell therapies, more attention is turning to the underlyingmanufacturing strategies that will allow these therapies to benefitpatients worldwide. While cell therapies hold great promise clinically,high manufacturing costs relative to reimbursement present a formidableroadblock to commercialization. Thus, the need for cost effectiveness,process efficiency and product consistency is driving efforts forautomation in numerous cell therapy fields, and particularly for T cellimmunotherapies (see, e.g., Wang 2016).

Recent successful clinical results from immunotherapy trials usingchimeric antigen receptor (CAR) T cells provide new hope to patientssuffering from previously untreatable cancers (see, e.g., Lu 2017;Berdeja 2017; Kebriaei 2016). As these novel therapeutics move from theclinical trial stage to commercial scale-up, challenges arise related tocell manufacturing (see, e.g., Morrissey 2017).

The production of these cells may require significant manual involvementdue to the patient-specific product. Automation of CAR T cell culture isparticularly challenging due to the multiple sensitive unit operations,including cell activation, transduction and expansion. Activation may beparticularly important as the efficiency of this process can impacttransduction and expansion.

Integration of cell activation, transduction and expansion into acommercial manufacturing platform is critical for the translation ofthese important immunotherapies to the broad patient population. Forthese life-saving treatments to be applicable to the global patientpopulation, a shift in manufacturing techniques must be implemented tosupport personalized medicine. The benefits of automation havepreviously been described (see, e.g., Trainor 2014; Mandavi 2015). Thesebenefits include labor time savings associated with using automation aswell as improved product consistency, decreased room classification,decreased clean room footprint, decreased training complexities, andimproved scale-up and tracking logistics. Furthermore, software can beused to streamline the documentation processes by using automaticallygenerated electronic batch records to provide a history of allprocessing equipment, reagents, patient identification, operatoridentification, in-process sensor data, and so forth.

SUMMARY OF THE INVENTION

In some embodiments provided herein is a method for automated productionof a genetically modified immune cell culture, the method comprising:activating an immune cell culture with an activation reagent to producean activated immune cell culture; transducing the activated immune cellculture with a vector, to produce a transduced immune cell culture;expanding the transduced immune cell culture; concentrating the expandedimmune cell culture of (c); and harvesting the concentrated immune cellculture of (d) to produce a genetically modified immune cell culture,further comprising washing either or both the expanded immune cellculture and the concentrated immune cell culture, wherein (a) through(e) are performed by a fully enclosed cell engineering system and (a)through (e) are optimized via a process to produce the geneticallymodified immune cell culture.

In further embodiments, provided herein is a method for promoting apreferred phenotype of a genetically modified immune cell culture, themethod comprising: activating an immune cell culture with an activationreagent to produce an activated immune cell culture, wherein theactivation reagent and activating conditions promote the phenotype ofthe genetically modified immune cell culture; transducing the activatedimmune cell culture with a vector, to produce a transduced immune cellculture; expanding the transduced immune cell culture; concentrating theexpanded immune cell culture of (c); and harvesting the concentratedimmune cell culture of (d) to produce a genetically modified immune cellculture, wherein (a) through (e) are performed by a fully enclosed,automated cell engineering system.

In additional embodiments, provided herein is a method for automatedproduction of a genetically modified immune cell culture, the methodcomprising: activating an immune cell culture with an activation reagentto produce an activated immune cell culture; transducing the activatedimmune cell culture with a vector, to produce a transduced immune cellculture; expanding the transduced immune cell culture; concentrating theexpanded immune cell culture of (c); and harvesting the concentratedimmune cell culture of (d) to produce a genetically modified immune cellculture, wherein (a) through (e) are performed by a fully enclosed,automated cell engineering system, and wherein each of (a) through (e)are performed with immune cell cultures having an optimized cell density(cells/mL) and an optimized cell confluency (cells/cm²).

In additional embodiments, provided herein is a method for automatedproduction of a genetically modified immune cell culture, the methodcomprising: activating an immune cell culture with an activation reagentto produce an activated immune cell culture; transducing the activatedimmune cell culture with a vector, to produce a transduced immune cellculture; expanding the transduced immune cell culture, wherein thetransduced cell culture is not shaken during the expanding;concentrating the expanded immune cell culture of (c); and harvestingthe concentrated immune cell culture of (d) to produce a geneticallymodified immune cell culture, wherein (a) through (e) are performed by afully enclosed, automated cell engineering system.

In still further embodiments, provided herein is a method for automatedproduction of a genetically modified immune cell culture, the methodperformed by a cell engineering system, comprising: activating an immunecell culture with an activation reagent to produce an activated immunecell culture in a first chamber of the cell engineering system;transducing the activated immune cell culture, the transducingcomprising: transferring the activated immune cell culture from thefirst chamber to an electroporation unit; electroporating the activatedimmune cell culture with a vector, to produce a transduced immune cellculture; transferring the transduced immune cell culture to a secondchamber of the cell engineering system; expanding the transduced immunecell culture; concentrating the expanded immune cell culture of (c); andharvesting the concentrated immune cell culture of (d) to produce agenetically modified cell culture.

In additional embodiments, provided herein is a cassette for use in anautomated cell engineering system, comprising: a low temperaturechamber, for storage of a cell culture media; a high temperature chamberfor carrying out activation, transduction and expansion of an immunecell culture, wherein the high temperature chamber is separated from thelow temperature chamber, by a thermal barrier, the high temperaturechamber including a cell culture chamber; and one or more fluidicspathways connected to the cell culture chamber, wherein the fluidicspathways provide recirculation, removal of waste and homogenous gasexchange and distribution of nutrients to the cell culture chamberwithout disturbing cells within the cell culture chamber.

In still further embodiments, provided herein is a cassette for use inan automated cell engineering system, comprising: a cell culture chamberfor carrying out activation, transduction and/or expansion of an immunecell culture having a chamber volume that is configured to house animmune cell culture, a satellite volume for increasing the workingvolume of the chamber by providing additional volume for media and otherworking fluids without housing the immune cell culture, wherein thesatellite volume is fluidly connected to the cell culture chamber viaone or more fluidics pathways such that media is exchanged with theculture chamber without disturbing the immune cell culture.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a generalized manufacturing process for chimeric antigenreceptor (CAR) T cells.

FIG. 2 shows a lab space containing exemplary cell engineering systemsas described in embodiments herein.

FIG. 3 shows a CAR T cell production process that can be performed in acell engineering system as described in embodiments herein.

FIG. 4 shows comparisons between the COCOON system and control methodsfor maintaining populations of CD8+ and CD4+ cells.

FIG. 5 shows comparisons between the COCOON system and control methodsfor amount of CAR T cells in the CD8+ and CD4+ cell populations.

FIGS. 6A-6C show an overview of a COCOON system as used in Example 1.

FIG. 6A shows a COCOON system in the closed configuration. FIG. 6B showsa Cassette that can be inserted into the COCOON. FIG. 6C shows a COCOONsystem in the open configuration.

FIGS. 6D-6E show the location and orientation of a cell culture chamberutilized in a COCOON system.

FIG. 6F shows a more detailed view of the cell culture chamber utilizedin a COCOON system.

FIG. 6G shows process flow legend for a COCOON system.

FIG. 6H shows gas transfer data using the COCOON system.

FIGS. 7A-7C show results of experiments described in Example 1,comparing GFP transduction in the COCOON system and manual manipulation.FIG. 7A shows a comparison of average harvest yields. FIG. 7B shows acomparison of average harvest viability. FIG. 7C shows a comparison ofaverage transduction efficiency.

FIGS. 8A-8B show results of experiments described in Example 1,comparing HER-2 CAR T transduction in the COCOON system and PERMALIFEbag. FIG. 8A shows a comparison of the viable cell yield. FIG. 8B showsa comparison of viability and transduction efficiency.

FIGS. 9A-9D show results of experiments described in Example 1,comparing the COCOON system and PERMALIFE bag. FIG. 9A shows acomparison of relative CAR T purity. FIG. 9B shows a comparison of CD8+cell percentage. FIGS. 9C and 9D show production of TNFα and INFγ,respectively.

FIGS. 10A-10B show results of experiments described in Example 1,comparing the killing of target tumor cells by CAR T cells cultured inthe COCOON system (FIG. 10A) and the PERMALIFE bag (FIG. 10B).

FIGS. 11A-11E show another configuration of a COCOON system as describedin embodiments herein. FIG. 11A shows a disposable T cell cassette thatcan be loaded into the COCOON system. FIG. 11B shows a COCOON system inthe open configuration.

FIG. 11C shows the cassette loaded into the COCOON. FIG. 11D shows theCOCOON in a closed configuration. FIG. 11E shows a detailed view of acassette for use with the COCOON.

FIG. 11F shows the use of a syringe and a bag to sample from thecassette.

FIG. 12A shows a process overview for the CAR T cell production process.FIG. 12B shows a COCOON cassette cell proliferation chamber with a CARTcell culture in progress. FIG. 12C shows a manually manipulated CAR Tcell production process using a cell culture bag in an incubator.

FIGS. 13A-13H show results of experiments described in Example 2,comparing the PERMALIFE bag and COCOON system, as well as T cellactivation by DYNABEADS or OKT3. FIG. 13A compares viable cell yield.FIG. 13B compares population doubling level (PDL). FIG. 13C comparesviable CD3+ T cell yield. FIG. 13D compares CD3+ cells PDL. FIG. 13Ecompares percentage of CD3+ subsets (CD4+ and CD8+). FIG. 13F comparescell exhaustion as measured by anti-PD-1. FIGS. 13G and 13H showcytometry plots of CD8+CD3+ T cells activated with DYNABEADS or OKT3,respectively.

FIGS. 14A-14F show results of experiments described in Example 2,comparing the PERMALIFE bag and COCOON system. FIG. 14A comparestransduction efficiency of CD3+ cells. FIG. 14B compares total number ofviable CAR T cells. FIG. 14C compares transduction efficiency of T cellsubsets (CD4+ and CD8+). FIG. 14D compares total CAR T cells by subsets.FIGS. 14E and 14F show cytometry plots of CD3+ OKT3 activated cells inCOCOON and PERMALIFE bags, respectively.

FIGS. 15A-15F show results of experiments described in Example 2,comparing the PERMALIFE bag and COCOON system. FIG. 15A comparespercentage of cells producing TNFα. FIG. 15B compares percentage ofcells producing IFNγ. FIGS. 15C and 15D show cytometry plots ofDYNABEAD-activated COCOON-produced cells secreting TNFα and IFNγ,respectively. FIGS. 15E and 15F show tumor killing efficiency of CAR Tcells produced from PERMALIFE bags or COCOON system, respectively.

FIG. 16 shows a summary of the comparison between COCOON and PERMALIFE,and activation by DYNABEADS or OKT3.

FIG. 17 shows the incorporation of an electroporation unit with a cellengineering system, in accordance with embodiments hereof.

FIG. 18 shows the flow of immune cell culture from a cell engineeringsystem to an electroporation unit and back again.

FIG. 19 shows the results of cell expansion experiments.

FIG. 20 shows the results of cell expansion following 12 days ofexpansion.

FIG. 21 shows differentiation of cell phenotype.

FIG. 22 shows further evidence of differentiation of cell phenotype.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides an automated method of producingchimeric antigen receptor T (CAR T) cells. The production of CAR T cellstypically requires manual involvement due to the patient-specificproduct. Automation of CAR T cell culture has been particularlychallenging due to the multiple sensitive unit operations, includingcell activation, transduction, and expansion. Thus, disclosed herein areautomated methods of CAR T cell production utilizing a fully-enclosedcell engineering system.

Automated Cell Processing

For autologous cell treatments such as T cell therapy, the need for costeffectiveness, process efficiency, and product consistency isparticularly acute, as manufacturing micro-lot (one patient per lot)batches lacks the economies of scale that allogeneic (multiple patientsper lot) processes can exploit (see, e.g., Jones 2012; Trainor 2014).The larger and more localized workforce and facilities required formicro-lots places considerable demands on logistics, GMP compliance formanual production, especially with respect to availability and trainingof staff. In addition, the potential for variability in techniquebetween operators can pose an undesirable risk to consistently meetingrelease criteria and ensuring a safe and dependable product.

As described herein, installation and comprehensive validation ofautomated manufacturing provides a solution to these logistical andoperational challenges. An important approach to introducing automationto a production process is identifying the key modular steps where theoperator applies a physical or chemical change to the productionmaterial, termed “unit operations.” In the case of cell manufacturing,this includes steps such as cell separation, genetic manipulation,proliferation, washing, concentration, and cell harvesting.Manufacturers often identify focal process bottlenecks as the immediateopportunities for introducing automation. This is reflected in thetechnical operation spectrum of the majority of commercially availablebioreactors, which tend to focus on discrete process steps. Processchallenges in cell manufacturing (from sterility maintenance to sampletracking) are addressed herein by end-to-end automation that generatesconsistent cellular outputs while ameliorating inevitable processvariability. The methods described herein also provide simplification,and the associated electronic records aid in complying with GMPstandards (see, e.g., Trainor 2014).

Automation of Unit Operations and Key Process Sensitivities

The recent rapid progress of the clinical development of modifiedautologous T cells for cancer immunotherapy has led to planning for theassociated translation and scale up/out implications.

While specific protocols may vary for T cell manufacturing, ageneralized chimeric antigen receptor T cell (CAR T) process isillustrated in FIG. 1. FIG. 1 describes unit operations of CAR T cellmanufacturing, from initial processing of a patient blood sample toformulating output cells for autologous T cell therapy.

As described herein, to achieve cell manufacturing automation, themethods described herein provide for understanding the status of thecells at each transition point and how they are impacted by the specificunit operation. The micro-lot production for patient-specific therapiesshould be respectful of key process sensitivities that impact thefeasibility of automation. Automation described herein successfullyembraces various process steps.

Table 1 below highlights the challenges of some process steps identifiedfor T cell automation and notes the impact of the sensitivity on theautomation strategy. Note that for all unit operations, open transfer ofcells between respective equipment is a key sensitivity due to the riskof contamination.

TABLE 1 Automation Challenges and Benefits Challenges of Key UnitOperation Process Steps Benefit of Automating Fractionation Highlyvariable based on High purity of target starting donor cells andoperator population technique (see e.g., More consistent and improvedNilsson 2008) product Residual impurities can impact performance CellSeeding Inhomogeneous cell Homogenous automated distribution leads toseeding strategy can improve variability in growth rates consistency andpotency Activation Stable contact between Automated loading can ensurecells and activation reproducibly homogeneous reagent distribution andactivation Uniform activation - which can be difficult to homogeneousconsistently achieve with distribution manual methods TransductionEfficiency can be Volume reduction prior to virus affected by the degreeof addition enables high degree of cell-virus mixing, which cell-viruscontact may vary based on Time-based operation enables operator handlingcell transfer regardless of time Increased exposure time of day may havenegative Closed system decreases risk impact on cells to operatorElectroporation Efficiency can vary Standardized protocols ensure basedon operator consistent results when mixing, washing and upstream anddownstream concentration technique steps are integrated Feeding Timingof media Biofeedback can optimize exchange needs to feeding schedule(see, e.g., Lu consider nutritional 2013) and minimize media userequirements based on Components can be stored at cell growth (see,e.g., refrigerated temperatures to Bohenkamp 2002), and prolongstability and the component stability automatically pre-warmed at 37° C.before use Selection Extensive handling steps Full automation improvescan result in cell loss consistency Operator variability HarvestAcellular materials (such Cells automatically transferred as cellseparation beads) from culture vessel regardless to be removed prior toof time of day final formulation (see Improved final yield e.g.,Hollyman 2009) consistency over manual Manual pipetting pipettingvariability can impact final yield Washing Aggressive washing may Gentlewashing, filtration, or induce shear stress or sedimentation withoutmoving cause cell loss during the culture vessels, can be supernatantremoval utilized to reduce cell loss and remove residuals ConcentrationCell recovery may vary Automated volume reduction by operator duringreduces operator variability aspiration Filtration methods also minimizecell loss Formulation Product must be well Automated mixing ensuresmixed homogenous distribution of Small working volumes cells in finalformulation magnify impact of Automated volume addition volumeinaccuracies removes risk of manual Viability decreases with pipettingerror or variability longer exposure times to Increased automationreduces cryopreservative variability in temperature sensitive steps

Tailoring the automation of a manual process around the sensitivitieslisted in Table 1 can support successful translation, maintenance orimprovement on the performance of the cell therapy.

Integration of Automated Unit Operations

Along with considering the GMP logistics, economics and patient safetyimplications of automation, unit operations can be assessed in thecontext of typical labor hours per unit operation (including workinghours for both the operator and the quality assurance monitor). Table 2identifies nominal manual processing timelines for representative stepsin CAR T automation. This table highlights the resource commitmentsrequired for each unit operation in a generalized CAR T cell process.For each step, the estimated remaining labor time for an automatedprocess is identified, as well as the rationale for the reduction.

TABLE 2 Automation Reduction of Labor Hours Manual Automated UnitOperation Labor Labor Labor Reduction Incoming 2 hours 0.5 hoursIdentification, sample tracking details Documentation and operation logall initiated by uniform labelling and corresponding software Reagent 4hours 2 hours Single reagent preparation step with Preparation storagein a refrigerated zone removes need to prepare reagents before each unitoperation Isolation from 3 hours 0.5 hours Once blood sample is loaded,whole blood automated PBMC isolation from whole blood possible usingcentrifugation (see, e.g., FDA 2011), filtration (see, e.g., Wegener2014) and/or antibody selection Cell Seeding 1 hour 0 hours Automatedseeding immediately after fractionation Activation 2 hours 0 hours Tcell activation by common methods such as antibodies or beads performedby automated mixing of reagents with cell culture (see, e.g., Trickett2003) Activation by dendritic cell co-culture would invoke the sameautomated culture principles (see, e.g., Hasegawa 2006) Transduction 6hours 2 hours T cells automatically transferred to a transductionchamber (with optional coating if viral vectors used) Manual interactionrequired to attach viral vectors if not stable in refrigeratedconditions Electroporation 2 hours 0 hours Integrated electroporationremoves the need for additional preparation steps Cell Feeding 13.5hours 0 hours Media removal and feeding automated Washing 1 hour 0 hoursAutomated and integrated gentle cell washing Cell concentration byfiltration reduces time spent washing compared with centrifugationIn-Process 2 hours 1 hours Biosensor monitoring (e.g. pH, oxygen,Documentation/ glucose) Monitoring Responses to process readouts pre-programmed; potentially averting emergencies Application of imagingtechnology to processes such as fluid monitoring (see, e.g., Odeleye2014) and cell counting (see, e.g., Grishagin) being developed forautomated processes Selection 2 hours 0 hours Automated mixing of cellsand selection reagents Magnetic cell sorting performed by bindingantibody-conjugated beads to cells and passing them through a magnetizedchamber Concentration 2 hours 0 hours Cell centrifugation or filtrationall automated Harvest 2 hours 0 hours T cells automatically harvested byagitation, fluid flow and washing Release Testing 9 hours 7 hoursBiomass or capacitance detection indicate relative abundance of cellsAutomated cell counters, flow cytometers, and other analysis equipmentreduce manual counting time Phenotypic and functional assays stilllikely require manual labor Final 2 hours 1 hour Cell concentration andmixing with Formulation or formulation solution automatedCryopreservation Notifications to operator required for quick transferto controlled rate freeze if not being shipped Outgoing 4 hours 2 hoursIdentification, sample tracking details Tracking and and operation logall generated by Documentation software for labelling and delivery topatient Total Labor 57.5 hours 16 hours Automation can lead to a 72%reduction in labor time

Based on the methods described herein, the automation of unit operationscan reduce a nominal manual process by nearly 40 hours to approximatelya quarter of the original time.

Discrete versus Fully Integrated Automation

While there is compelling evidence for the value of automation (see,e.g., Trainor 2014; Levine 2017), there needs to be a subsequentanalysis on the value and practicality of integrating these automationsteps in an end-to end sequence with automated transfers. There aredifferent perspectives on the advantages of discrete process automationversus the advantages of end-to-end integration.

The key benefit to discrete automation is flexibility. This relates tothe areas of:

-   -   1) Maintenance of unique process operations    -   2) Acceleration of translational activities based on individual        unit operation validation    -   3) Ability to modify processing steps to accommodate        donor-to-donor variability

The first point related to increased flexibility provides the operatorwith more control of the process. This is important in circumstanceswhere the process has highly sensitive steps that can impact the finalproduct. Switching to an all-in-one system may impose constraints thatinfluence the product outcome. A discrete approach provides theflexibility to choose how to perform each step, which may beparticularly important with highly sensitive unit operations. Thediscrete approach also allows gradual translation into automation frommanual processing, which helps to demonstrate equivalency if each unitoperation can be tested independently. Additionally, automating specificunit operations provides the flexibility for decisions to be made basedon the cell performance. For example, if cells are growing rapidly,there may be the need to expand from one cell culture bag to two.Lastly, the approach to automation using discrete systems also enablesgroups to pick-and-choose which equipment to use for each unitoperation.

Equipment utilization is another argument for discrete automation. Theremay be some unit operations that require significantly more time thanothers. An end-to-end processing system requires all multiple unitoperations to run on a single system, thus occupying the equipment forthe duration of the culture process.

While there are benefits to discrete automation, an end-to-end approachoffers different, though no less compelling benefits. Firstly, a fullyintegrated system greatly reduces the risk of contamination. As there isincreased handling required with a discrete approach, there is a greaterchance of product variability due to operator interventions. Secondly,and as previously mentioned, this inevitably leads to higher laborcosts.

The flexibility provided by the discrete approach is important. Insituations where the process is important in defining the product, anend-to-end system should have the flexibility to integrate uniquesensitivities. This may include certain feeding strategies, oxygenlevels, surface treatments, and so forth. Such an approach requiresflexibility in both the software and the disposable component. Thesystem should provide the option to pull cell and media samples atvarious points in the process to confirm that specific unit operationsmeet product specification checkpoints. If modifications need to bemade, the software should be able to implement these changes to provideideal conditions. While easy-to-use and flexible software is highlybeneficial for translational purposes, it is important that the softwarecan be easily locked down to comply with clinical standards (FDA 21 CFRPart 11). Once locked down, there should be limited if any ability forthe operator to change the protocol. However, to address issues withinherent donor-variability, there should be the option to select from arange of validated protocols based on cell growth rates. For example, ifthe cells are growing rapidly, the system should be able to respond tothis and adjust the feed or harvest time points, accordingly.

The selection of end-to-end integration versus discrete automation isalso dependent upon the long-range vision for the clinical process. Asingle all-in-one system can offer significantly greater spaceefficiency to minimize the required footprint in expensive GMP cleanrooms. For example, as shown in FIG. 2, fully integrated automatedsystems are designed to maximize required footprint to reduce expensiveGMP clean room space. FIG. 2 shows 96 patient-specific end-to-end unitsrunning in a standard lab space.

A single system also provides greater ease of data tracking, whereasdiscrete systems may not offer compliant software that links togetherall electronic data files. Software platforms such as VINETI (VinetiLtd) and TRAKCEL (TrakCel Ltd) allow electronic monitoring andorganization of supply chain logistics. However, single all-in-oneculture systems can go further still by incorporating a history of bothprocessing events and biomonitoring culture conditions associated witheach unit operation into a batch record. Accordingly, the benefits ofend-to-end integration offer a significant competitive advantage.

Commercial Platforms for Integration of Unit Operations

Clinical trial success in a number of autologous cell therapies,especially immunotherapy for blood-based cancers, has highlighted theimportance of enabling translation of new clinical protocols to robustproduction platforms to meet projected clinical demand (see, e.g.,Levine 2017; Locke 2017). For autologous therapies, processing eachpatient-specific cell treatment suitably utilizes comprehensivemanufacturing activities and operations management. The methods hereinlink unit operations in a turnkey automated system to achieve processoptimization, security and economy.

The challenge in designing an autologous process is two-fold. Firstly,unlike allogeneic manufacturing in which separate processing steps canoccur in physically separate and optimized pieces of equipment,scaled-out autologous platforms suitably perform all of the necessarysteps in a single closed, self-contained automated environment.Secondly, unlike an allogeneic process in which every run theoreticallystarts with a high-quality vial from a cell bank, with known quality andpredictable process behavior, the starting material in an autologousprocess is highly variable, and generally comes from individuals withcompromised health.

Thus, provided herein are methods that are able to sense cultureconditions and respond accordingly as a sophisticated bioreactor, bycontrolling factors such as physical agitation, pH, feeding, and gashandling. Furthermore, there are significantly different challenges withtechnology transfer related to autologous treatments compared toallogeneic treatments. Autologous products may have greater restrictionson stability between the manufacturing process and the patienttreatment. Sites can be located globally rather than at a single center.Having a locked down (e.g., fully enclosed) all-in-one systemsignificantly improves the technology transfer process between sites.

While source variability cannot be eliminated, automation helps toremove variability of the final autologous product throughstandardization and reproducibility. This practice is adopted by leadingcell system providers to obtain a cell performance reference point viabiosensors that monitor the status of the active cell cultures. Inend-to-end integration, output from any specific stage in the processshould be within acceptable parameters for the onward progression of theprocess.

As described herein, in embodiments, the methods provided utilize theCOCOON platform (Octane Biotech (Kingston, ON)), which integratesmultiple unit operations in a single turnkey platform. Multiple cellprotocols are provided with very specific cell processing objectives. Toprovide efficient and effective automation translation, the methodsdescribed utilize the concept of application-specific/sponsor-specificdisposable cassettes that combine multiple unit operations—all focusedon the core requirements of the final cell therapy product.

The methods described herein have been used to expand CAR T cells(including activation, viral transduction and expansion, concentrationand washing) in a fully-integrated closed automation system (FIG. 3).

In the experiments conducted, the fold expansion of CAR T cells, in10-14 day cultures, reached around 40 to 60. Both CD4+ and CD8+ T cellsubsets are required for successful CAR T therapy. Therefore, the runsand associated controls were evaluated via flow cytometry for theirability to maintain cultures of both T cell subsets. FIG. 4 shows thatall runs as well as all controls were able to maintain both T cellsubsets. The percentage of CAR T cells present was also evaluated ineach population of T cell subset (FIG. 5). In all samples, there was ahigher detection of NGFR (indicative of CAR construct) in the CD4+fraction compared to the CD8+ fraction although in all samples, theNGFR+ fraction in the CD8+ portion was >50% of the fraction found in thepaired CD4+ population. In summary, automated CAR T process using themethods described herein yields healthy populations of T cell subsets.

Advantages of Automation

Automation of unit operations in cell therapy production provides theopportunity for universal benefits across allogeneic and autologous celltherapy applications. In the unique scenario of patient-specific,autologous cell products, and ever more emphasized by the recentclinical success of these therapies, the advantages of automation areparticularly compelling due to the significant micro-lot complexities ofsmall batch GMP compliance, economics, patient traceability and earlyidentification of process deviations. The associated emergence ofcomplex manufacturing protocols draws attention to the fact that thevalue of end-to-end integration of automated unit operations inmicro-lot cell production has not been a point of significant study.However, the expected demand for these therapies following theirimpending approval indicates that implementation of a fully closedend-to-end system can provide a much needed solution to manufacturingbottlenecks, such as hands-on-time and footprint.

Developers of Advanced Therapies are encouraged to consider automationearly in the rollout of clinical translation and scale up of clinicaltrial protocols. Early automation can influence protocol development,avoid the need for comparability studies if switching from a manualprocess to an automated process at a later stage, and provide a greaterunderstanding of the longer-term commercialization route.

Methods of Producing Genetically Modified Immune Cells, Including CAR TCells

In embodiments, provided herein is a method for automated production ofa genetically modified immune cell culture. As used herein a“genetically modified immune cell culture” (or genetically modifiedimmune cells) refers to cells of the immune system that are modified orprimed (e.g., through co-culture with antigen presenting cells),resulting in cells that have a desired phenotype useful in treating,preventing or ameliorating one or more diseases in an animal, includinga human. As used herein an “immune cell culture” refers to a collectionof cells prepared by a method described herein, and can include a cellpopulation for use in research or clinical trials, as well as foradministration to a mammal, including a human patient, for a medicaltherapy. The genetically modified immune cell cultures that can beproduced using the methods described herein can include mast cells,dendritic cells, naturally killer cells, B cell, T cells, etc.

The various methods described herein can also be extended to othergenetically modified cell cultures, including for example, thegeneration of genetically modified human stem cell cultures, includinghematopoietic stem cells.

In exemplary embodiments, the method comprises activating an immune cellculture with an activation reagent to produce an activated immune cellculture, transducing the activated immune cell culture with a vector, toproduce a transduced immune cell culture, expanding the transducedimmune cell culture, concentrating the expanded immune cell culture andharvesting the concentrated immune cell culture to produce a geneticallymodified immune cell culture. Suitably, the method further includeseither or both the expanded immune cell culture and the concentratedimmune cell culture. In embodiments, the various steps of the method areperformed by a fully enclosed cell engineering system and are optimizedvia a process to produce the genetically modified immune cell culture.

Methods for optimizing the process for producing the geneticallymodified immune cells include optimization of cell culture conditionsbefore beginning an automated method, as well as the use of feedbackfrom various sensors, etc., to assist with real-time modifications togrowth conditions (e.g., gas concentration, media conditions,temperature, pH, waste and nutrient concentrations, etc.).

In embodiments, the optimizing process is a self-adjusting process, thatis one that does not require input from an external (human) user, and isable via various computer programs and conditions to determine therequired modifications to a cell culture or other characteristics tooptimize the automated process. In embodiments, the self-adjustingprocess includes monitoring with one or more of a temperature sensor, apH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor,and an optical density sensor. As described herein, the use of thesevarious sensors in the fully enclosed cell engineering system occurs atvarious times and locations within the system, and work together inconcert to provide the optimization. For example, the self-adjustingprocess can adjust (e.g., raise or lower) one or more of a temperature,a pH level, a glucose level, an oxygen level, a carbon dioxide level,and an optical density of the transduced T cell culture, based on themonitoring.

The optimization process can also be based on the unique characteristicsof the starting cell population, including for example, the total cellnumber, the source of the cells, the density of the cells, the age ofthe cells, etc. These starting cell population characteristics can beinput into a computer control system prior to beginning the automatedmethods, upon which the system will make various initial modificationsto optimize the methods, e.g., oxygen and carbon dioxide concentration,flow rates, incubation times, pH, etc. Alternately, the monitoring ofcell processes enables the automated characterization of the progress ofthe cell culture sequence from the starting population to enablecase-by-case adjustment of conditions for optimized final cell cultureproperties.

In exemplary embodiments, the methods described herein produce at leastabout 50 million viable genetically modified immune cells. In suitableembodiments, the methods described produce at least about 100 millionviable genetically modified immune cells, or at least about 200 millioncells, at least about 300 million cells, at least about 400 millioncells, at least about 500 million cells, at least about 600 millioncells, at least about 700 million cells, at least about 800 millioncells, at least about 1 billion cells, at least about 1.1 billion cells,at least about 1.2 billion cells, at least about 1.3 billion cells, atleast about 1.4 billion cells, at least about 1.5 billion cells, atleast about 1.6 billion cells, at least about 1.7 billion cells, atleast about 1.8 billion cells, at least about 1.9 billion cells, atleast about 2 billion cells, least about 2.1 billion, at least about 2.2billion, at least about 2.3 billion, at least about 2.4 billion, atleast about 2.5 billion, at least about 2.6 billion, at least about 2.7billion, at least about 2.8 billion, at least about 2.9 billion, or atleast about 3.0 billion genetically modified immune cells.

As described herein, the genetically modified immune cell cultureproduced by the methods is suitably a T cell culture, including achimeric antigen receptor T (CAR T) cell culture. In such embodiments,the vector utilized to produce such CAR T cells is a vector encoding achimeric antigen receptor. Suitably the immune cell culture comprisesperipheral blood mononuclear cells and/or purified T cells. Inembodiments, the immune cell culture comprises at least one accessorycell, suitably a monocyte or a monocyte-derived cell. As describedherein, in embodiments, the accessory cell comprises antigens for a Tcell receptor, including CD28, CD40, CD2, CD40L and/or ICOS.

Suitably, the activation reagent comprises an antibody or a dendriticcell. In embodiments, the antibody is immobilized on a surface, whichcan include an polystyrene plastic, silicone or other surface, includingfor example, the surface of a bead.

In other embodiments, the activation reagent comprises an antibody thatis a soluble antibody, including at least one of an anti-CD3 antibodyand an anti-CD28 antibody. Exemplary antibodies include OKT3.

Various methods for transducing the cells can be utilized in theautomated methods, including for example, viral infection,electroporation, membrane disruption, or combinations thereof.

In exemplary embodiments, the vector that is utilized in the methods isa lentiviral vector or a retrovirus. Suitably, the transducing comprisesmixing the vector in cell culture media and delivering the vector in themedia uniformly to the activated immune cell culture. As describedherein, the uniform delivery of the vector in a homogenous manner to thecells provides for optimization of the various cell characteristics ofhigh output of desired genetically modified immune cells.

As described herein, the methods of expanding the cells suitably includeat least one or more of feeding, washing, monitoring, and selecting ofthe transduced immune cell culture.

The various methods described herein are conducted in a manner such thatthe oxygen level of the transduced immune cell culture is optimized forthe immune cell culture. This optimization allows for production of alarge number of viable cells having the desired phenotypiccharacteristics, including, as described herein, the promoting of adesired cell phenotype. In embodiments, oxygen level or concentration isoptimized by the cell engineering system recirculating cell culturemedia through an oxygenation component during one or more of steps (a)to (e). As described herein, oxygenation suitably occurs through one ormore fluidic pathways, including silicone-based tubing components.

In further embodiments, the cell engineering system recirculatesnutrients, waste, released cytokines, and/or dissolved gasses during thevarious method processes. This recirculation helps aid in the productionof a large number of viable cells having the desired phenotype(s).Suitably, the carbon dioxide level provided by the cell engineeringsystem decreases during step the expansion step so as to optimize cellgrowth, etc. In other embodiments, the CO₂ level can be raised, forexample, if a complete media exchange is utilized.

Other mechanisms for optimizing the growth conditions for the cellsinclude modifying and controlling the flow rate of the media provided tothe cells. As the cells begin to grow, the circulation rate of the mediaprovided is increased, which improves gas exchange and allows oxygen andcarbon dioxide to either enter or leave the cell culture, depending onthe conditions of the cells and the requirements at the time.

In embodiments, the cell engineering system is configured to performseveral rounds of one or more of feeding, washing and monitoring, and inembodiments, selecting of the transduced immune cell culture. Thesevarious activities can be performed in any order, and can be performedalone or in combination with another activity. In embodiments,concentrating of the cells comprises centrifugation, supernatant removalfollowing sedimentation, or filtration. Suitably, the optimizationprocess further includes adjusting parameters of the centrifugation orfiltration, suitably in a self-adjusting process. Selecting of thetransduced cells can be carried out by, for example, magneticseparation, filtration, adherence to a plastic or other substrate, etc.

In embodiments as described herein, the cell engineering systemcomprises a plurality of chambers, and wherein each of the steps of themethod is performed in a different chamber of the plurality of chambersof the cell engineering system.

Suitably, the method further includes removing the activation reagentfrom the activated immune cell culture after step (a), and can includeremoving the vector following the transducing step. The activationreagent is suitably removed from the immune cell culture by washing,draining or physically removing the cells or the activation reagent. Thevector can be removed by washing, or by binding the vector to a surface(e.g., a retronectin or fibronectin coated surface) and thentransferring the cells to a different chamber.

In exemplary embodiments, the cell engineering system contains the cellculture, the activation reagent, the vector, and cell culture mediumprior to starting the method. In other embodiments, the activationreagent and/or the vector can be added separately following the start ofthe method of production, or at any suitable time during the process.

In additional embodiments, provided herein is method for promoting apreferred phenotype of a genetically modified immune cell culture, themethod comprising activating an immune cell culture with an activationreagent to produce an activated immune cell culture, wherein theactivation reagent and activating conditions promote the phenotype ofthe genetically modified immune cell culture, transducing the activatedimmune cell culture with a vector, to produce a transduced immune cellculture, expanding the transduced immune cell culture, concentrating theexpanded immune cell culture, and harvesting the concentrated immunecell culture of (d) to produce a genetically modified immune cellculture. As described herein, the methods are suitably performed by afully enclosed, automated cell engineering system.

As described herein, selection of the appropriate activation reagent andthe appropriate activation conditions provide for the promotion of adesired phenotype of a genetically modified immune cell culture. Thatis, the phenotype of the immune cell culture can be specificallyselected and promoted, so that suitable a majority of the cells that areproduced by the methods have the desired, preferred phenotype. In otherembodiments, a desired ratio of one cell phenotype to another phenotypecan be controlled and promoted, providing a desired, preferred phenotypebalance.

As described herein, it has been found that through the use ofactivation reagents that are antibodies, and particularly solubleantibodies, the desired phenotype of a genetically modified immune cellcan be promoted. Suitably, the antibodies that are utilized are at leastone of an anti-CD3 antibody, an anti-CD28 antibody and an anti-CD2antibody, including the soluble antibody OKT3.

In embodiments, the activating conditions provide a substantiallyundisturbed immune cell culture allowing for stable contact between theactivation reagent and the immune cell culture. As described herein, ithas been found that allowing the cells to activate under substantiallyundisturbed conditions, and via the use of a cell culture chamber thatis flat and substantially non-flexible. This provides an environmentwhere the cells can be homogenously contacted with the activationreagent, as well as interact with the necessary nutrients, dissolvedgasses, etc., to achieve the desired and promoted phenotype.

The methods described herein can influence the characteristics of thefinal immune cell culture product by selecting an appropriate activationmethod to provide the preferred phenotype. For example, activationutilizing a bead-based process as described herein promotes a morebalanced CD4:CD8 ratio, whereas use of a soluble anti-CD3 promotes ahigher population of CD8 than CD4. Other levels of CD8 and CD4 can alsobe provided using the methods described herein. In exemplaryembodiments, as described herein, the methods can be utilized to prepareCAR T cells. Suitably, the methods can be utilized to promote aphenotype of the CAR T cells that has a ratio of CD8+ cells to CD4+ ofabout 0.1:1 to about 10:1, including a ratio of CD8+ cells to CD4+ cellsof about 0.5:1 to about 5:1, about 0.8: to about 3:1, or about 1:1,about 2:1, etc.

In additional embodiments, methods are provided for automated productionof a genetically modified immune cell culture, the method comprising,activating an immune cell culture with an activation reagent to producean activated immune cell culture, transducing the activated immune cellculture with a vector, to produce a transduced immune cell culture,expanding the transduced immune cell culture, concentrating the expandedimmune cell culture of (c), and harvesting the concentrated immune cellculture of (d) to produce a genetically modified immune cell culture. Asdescribed herein, the method is suitably performed by a fully enclosed,automated cell engineering system. In embodiments, each of the steps ofthe method is performed with immune cell cultures having an optimizedcell density (cells/mL) and an optimized cell confluency (cells/cm²).

As described herein, it has been determined that utilizing an optimizedcell density (cells per mL of cell media) and/or cell confluency (cellsper area (cm²) of a cell culture chamber on which the cells are beingacted one and grown), provide for increased production of viable cells,as well as better control of cell phenotype, etc.

In embodiments, the optimized cell density for is about 0.05*10⁶cells/mL to about 60*10⁶ cells/mL, about 0.05*10⁶ cells/mL to about40*10⁶ cells/mL, or about 0.05*10⁶ cells/mL to about 20*10⁶ cells/mL.The optimized cell density can vary over the course of the methods ofproduction, such that at each stage of the method (i.e., activating,transducing, expanding, concentrating), the cell density is controlledor manipulated to provide the best cell density for that particular stepof the method. The cell density can be optimized by, for example,selection of the optimal starting cell density, increasing or decreasingoxygen and/or carbon dioxide concentration, regulating pH, temperature,nutrients, removal of waste, etc. Exemplary cell densities include about0.05*10⁶ cells/mL, about 0.08*10⁶ cells/mL, about 1*10⁶ cells/mL, about5*10⁶ cells/mL, about 10*10⁶ cells/mL, about 20*10⁶ cells/mL, about30*10⁶ cells/mL, about 40*10⁶ cells/m L, about 50*10⁶ cells/mL, or about60*10⁶ cells/mL, etc.

In embodiments, the optimized cell confluency for is about 0.1*10⁶cells/cm² to about 60*10⁶ cells/cm², or about 0.1*10⁶ cells/cm² to about40*10⁶ cells/cm², or about 0.1*10⁶ cells/cm² to about 20*10⁶ cells/cm².The optimized cell confluency can vary over the course of the methods ofproduction, such that at each stage of the method (i.e., activating,transducing, expanding, concentrating), the cell confluency iscontrolled or manipulated to provide the best cell confluency for thatparticular step of the method. The cell confluency can be optimized by,for example, selection of the optimal starting cell confluency, materialselection of the cell culture chamber, increasing or decreasing oxygenand/or carbon dioxide concentration, regulating pH, temperature,nutrients, removal of waste, etc. Exemplary cell confluency includeabout 0.1*10⁶ cells/cm², about 0.5*10⁶ cells/cm², about 1*10⁶ cells/cm²,about 0.5*10⁶ cells/cm², about 10*10⁶ cells/cm², about 20*10⁶ cells/cm²,about 30*10⁶ cells/cm², about 40*10⁶ cells/cm², about 50*10⁶ cells/cm²,or about 60*10⁶ cells/cm², etc.

In embodiments, the methods include the recirculation of nutrients,waste, released cytokines, and/or dissolved gasses are homogenouslyprovided to the cells having a density of about 0.05*10⁶ cells/mL toabout 20*10⁶ cells/mL and a confluency of about 0.1*10⁶ cells/cm² toabout 20*10⁶ cells/cm².

In further embodiments, methods for automated production of agenetically modified immune cell culture are provided, the methodcomprising activating an immune cell culture with an activation reagentto produce an activated immune cell culture, transducing the activatedimmune cell culture with a vector, to produce a transduced immune cellculture, expanding the transduced immune cell culture, wherein thetransduced cell culture is not shaken during the expanding,concentrating the expanded immune cell culture, and harvesting theconcentrated immune cell culture of to produce a genetically modifiedimmune cell culture. As described herein, suitably the methods areperformed by a fully enclosed, automated cell engineering system.

As described herein, it has been surprisingly found that allowing thecells to expand under conditions where they are not shaken (i.e., notrotated or shaken in order to cause the cells to flow over top of oneanother), the methods provide optimal cell characteristics, includinghigh viable cell yield and desired phenotypes. It has been determinedthat a large, un-shaken cell culture chamber, can provide homogenousaccess of the cells to the necessary reagents, nutrients, gas exchange,etc., while removing cellular waste, without the requirement to shake ordisturb the cells to achieve the desired outcome. In fact, as describedherein, it has been found that such methods for the automated productionof genetically modified immune cells produce higher numbers of viablecells, greater numbers/ratios of desired cells types, and more robustcellular characteristics, as compared to methods that utilize cellularshaking, for example, as described in Miltenyi et al., “SampleProcessing System and Methods,” U.S. Pat. No. 8,727,132.

Suitably, the expanding step of the methods include at least one or moreof feeding, washing, monitoring, and selecting of the transduced immunecell culture, without shaking the immune cell culture.

Also provided herein are methods for automated production of agenetically modified immune cell culture, the method performed by a cellengineering system, comprising activating an immune cell culture with anactivation reagent to produce an activated immune cell culture in afirst chamber of the cell engineering system, transducing the activatedimmune cell culture. In exemplary methods, the transducing comprisestransferring the activated immune cell culture from the first chamber toan electroporation unit, electroporating the activated immune cellculture with a vector, to produce a transduced immune cell culture, andtransferring the transduced immune cell culture to a second chamber ofthe cell engineering system. The methods further include expanding thetransduced immune cell culture, concentrating the expanded immune cellculture of, and harvesting the concentrated immune cell culture of (d)to produce a genetically modified cell culture.

For example, as shown in FIG. 17, an activated immune cell culture istransferred, e.g., via connection tubing 1704, from cassette 602 of acell engineering system 600 to an electroporation unit 1706.Electroporation unit 1706 suitably includes an electroporation cartridge1708, which holds the cell culture during the electroporation process.Following the electroporation process, the transduced immune cellculture is transferred back, via connection tubing 1704, to cellengineering system 600. FIG. 17 also shows the use of two optionalreservoirs 1710 and 1712, which are used to hold the cell culture priorto and after electroporation, to help in the transfer between the cellengineering system and the electroporation unit as a result of differentpump speeds, required pressures and flow rates. However, such reservoirscan be removed and the cell culture transferred directly from cellengineering system 1702 to electroporation unit 1706.

FIG. 18 shows a flow diagram of the cell culture 1) from the cellengineering system to a first reservoir, 2) to the electroporation unit,3) to a second reservoir, and finally 4) back to cell engineeringsystem.

In exemplary embodiments, as shown in FIGS. 17 and 18, electroporationunit 1706 is located outside of cell engineering system 1702. In suchembodiments, the transducing comprises transferring via a first sterile,closed connection (e.g., connection tubing 1704), the activated immunecell culture from the first chamber to the electroporation unit,electroporating the activated immune cell culture with the vector, toproduce the transduced immune cell culture, and transferring via asecond sterile, closed connection (e.g., connection tubing 1704), thetransduced immune cell culture to the second chamber of the cellengineering system.

It should also be understood that multiple, separate cell engineeringsystems 600 (see, e.g., FIG. 2) can be connected to a singleelectroporation unit, and run in appropriate order such that cellcultures are transferred from the cell engineering systems, to theelectroporation unit, and then back to the appropriate cell engineeringsystem.

In other embodiments, electroporation unit 1706 can be located withincell engineering system 600, such that the entire system is a closed,self-contained system. Methods for including electroporation unit 1706inside of cell engineering system 600 are known by those of ordinaryskill in the art, and utilize various miniaturization strategies, etc.

The various methods described herein allow for the production ofgenetically modified immune cell cultures where the transductionefficiency of the method is at least 20% higher than the transductionefficiency of the method utilizing a flexible, gas permeable bag forcell culture. As described herein, and as demonstrated in the Examples,the methods utilizing a cell engineering system as described herein aresuperior to traditional methods which rely on the use of a flexible, gaspermeable bag for carrying out the cell culture. In further embodiments,the transduction efficiency of the method is at least 10% higher thanthe transduction efficiency of the method utilizing a flexible, gaspermeable bag for cell culture, more suitably at least 20% higher, atleast 25% higher, at least 30% higher, at least 35% higher, or inembodiments, at least 40% higher.

Suitably, the methods described herein produce at least 20% moregenetically modified immune cells than a method utilizing manual cellculture with a flexible, gas permeable bag. More suitably, the methodsproduce at least 25% more genetically modified immune cells, at least30% more genetically modified immune cells, at least 35% moregenetically modified immune cells, or at least 40% more geneticallymodified immune cells than a method utilizing manual cell culture with aflexible, gas permeable bag.

In exemplary embodiments, the cell engineering systems described hereincomprise a plurality of chambers, and wherein each of steps of thevarious method described herein are performed in a different chamber ofthe plurality of chambers of the cell engineering system, each of theactivation reagent, the vector, and cell culture medium are contained ina different chamber of the plurality of the chambers prior to startingthe method, and wherein at least one of the plurality of chambers ismaintained at a temperature for growing cells (e.g., at about 37° C.)and at least one of the plurality of chambers is maintained at arefrigerated temperature (e.g., at about 4-8° C.).

In some embodiments, the disclosure provides a method of producingchimeric antigen receptor T cells, the method including: (a) activatinga peripheral blood mononuclear cell culture, suitably with culture mediacomprising at least one of an anti-CD3 antibody and an anti-CD28antibody, to produce an activated T cell culture; (b) transducing theactivated T cell culture with a lentiviral vector, the vector encoding achimeric antigen receptor, to produce a transduced T cell culture; (c)expanding the transduced T cell culture to a pre-defined culture size;(d) concentrating the expanded T cell culture of (c) to a volume ofabout 20 mL to about 500 mL, suitably about 50 mL to about 200 mL; and(e) harvesting the concentrated T cell culture of (d) to produce achimeric antigen receptor T (CAR T) cell culture, wherein the activatedT cell culture is substantially undisturbed during steps (a) to (b);wherein the method is performed by a fully enclosed cell engineeringsystem, suitably having instructions thereon for performing steps (a) to(e). Suitably steps (a) to (e) are performed in one or more chambers ofthe cell engineering system. As described herein, in embodiments, themethod produces at least 20% more CAR T cells than a method utilizing aflexible, gas permeable bag for cell culture. In exemplary embodiments,the method produce at least 2 billion viable CAR T cells.

A chimeric antigen receptor T cell, or “CAR T cell,” is a T cell that ismodified with a chimeric antigen receptor (CAR) to more specificallytarget cancer cells. In general, a CAR includes three parts: theectodomain, the transmembrane domain, and the endodomain. The ectodomainis the region of the receptor that is exposed to extracellular fluid andincludes three parts: a signaling peptide, an antigen recognitionregion, and a spacer. The signaling peptide directs the nascent proteininto the endoplasmic reticulum. In CAR, the signaling peptide is asingle-chain variable fragment (scFv). The scFv includes a light chain(V_(L)) and a heavy chain (V_(H)) of immunoglobins connected with ashort linker peptide. In some embodiments, the linker includes glycineand serine. In some embodiments, the linker includes glutamate andlysine.

The transmembrane domain of the CAR is a hydrophobic α-helix that spansthe membrane. In some embodiments, the transmembrane domain of a CAR isa CD28 transmembrane domain. In some embodiments, the CD28 transmembranedomain results in a highly expressed CAR. In some embodiments, thetransmembrane domain of a CAR is a CD3-ζ transmembrane domain. In someembodiments, the CD3-ζ transmembrane domain results in a CAR that isincorporated into a native T cell receptor.

The endodomain of the CAR is generally considered the “functional” endof the receptor. After antigen recognition by the antigen recognitionregion of the ectodomain, the CARs cluster, and a signal is transmittedto the cell. In some embodiments, the endodomain is a CD3-ζ endodomain,which includes 3 immunoreceptor tyrosine-based activation motifs(ITAMs). In this case, the ITAMs transmit an activation signal to the Tcell after antigen binding, triggering a T cell immune response.

During production of CAR T cells, T cells are removed from a humansubject, genetically altered, and re-introduced into a patient to attackthe cancer cells. CAR T cells can be derived from either the patient'sown blood (autologous), or derived from another healthy donor(allogenic). In general, CAR T cells are developed to be specific to theantigen expressed on a tumor that is not expressed in healthy cells.

Activation of T Cells. In some embodiments, an immune cell cultureproduced by the methods described herein is a CAR T cell culture. CAR Tcells can be activated to form an activated T cell culture. In vivo,antigen-presenting cells (APCs), such as dendritic cells, act as thestimulus for T cell activation through the interaction of the T CellReceptor (TCR) with the APC major histone compatibility complex (MHC).TCR associates with CD3, a T cell co-receptor that helps to activateboth cytotoxic T cells (e.g., CD8+ naïve T cells) and T helper cells(e.g., CD4+ naïve T cells). In general, T cell activation follows atwo-signal model, requiring stimulation of the TCR/CD3 complex as wellas a co-stimulatory receptor. Activation of T cells is further describedin, e.g., Kochenderfer 2015; Kalos 2011.

Without the co-stimulatory signal, the cells are susceptible to anergyand become non-responsive. Thus, T cell co-stimulation may be importantfor T cell proliferation, differentiation, and survival. Non-limitingexamples of co-stimulatory molecules for T cells include CD28, which isa receptor for CD80 and CD86 on the membrane of APC; and CD278 or ICOS(Inducible T-cell COStimulator), which is a CD28 superfamily moleculeexpressed on activated T cells that interacts with ICOS-L. Thus, in someembodiments, the co-stimulatory molecule is CD28. In other embodiments,the co-stimulatory molecule is ICOS. In vivo, the co-stimulatory signalcan be provided by the B7 molecules on the APC, which bind to the CD28receptor on T cells. B7 is a peripheral transmembrane protein found onactivated APCs that can interact with CD28 or CD152 surface proteins ona T cell to produce a co-stimulatory signal. Thus, in some embodiments,the co-stimulatory molecule is B7. Co-stimulatory receptors are furtherdescribed in, e.g., Lafferty 1975; Harding 1992; Clavreul 2000; Charron2015; Fathman 2007; Greenwald 2005. Co-stimulation is further describedin, e.g., Carpenter 2000; Andris 2004. B7 molecules are furtherdescribed in, e.g., Fleischer 1996; Schwartz 2003.

Various methods of activation are utilized in vitro to simulate T cellactivation. In embodiments, a T cell culture is activated with anactivation reagent. In further embodiments, the activation reagent is anantigen-present cell (APC). In still further embodiments, the activationreagent is a dendritic cell. Dendritic cells are APCs that processantigen and present it on the cell surface to T cells. In someembodiments, the activation reagent is co-cultured with the T cellculture. Co-culturing may require separate purification and culturing ofa second cell type, which may increase labor requirements and sources ofvariability. Thus, in some embodiments, alternative activation methodsare used.

In embodiments, the cells maintain stable contact with the activationreagent during the activating step. One way to maintain stable contactbetween the cells and the activation reagent is by preventingunnecessary or excessive movement of the cells. Accordingly, inembodiments, the cell culture is substantially undisturbed during theactivation step. “Substantially undisturbed” means that the cellsgenerally remain in the same area of the cell culture chamber, e.g., thebottom of the chamber, while the cell culture media is being changed.Cells may be disturbed if they are moved between different vessels,e.g., transferred from one culture flask to another, or cells may bedisturbed if the vessel is flexible. A flexible vessel such as, e.g., aculture bag, can cause the cells to move when the bag is handled. Asdescribed herein, the methods suitably utilize a cell culture chamberthat is substantially flat, and low, to allow for uniform access of thecells to various nutrients and gases, also allowing for ease of removalof waste products and media transfer. The substantially flat cellculture chamber also allows for the cells to touch each other duringvarious stages of the methods which can enhance cell growth andproduction of the desired cell phenotype(s).

In some embodiments, the activation reagent is an antibody. In someembodiments, the cell culture is activated with an antibody bound to asurface, including a polymer surface, including a beads In furtherembodiments, the one or more antibodies is an anti-CD3 and/or anti-CD28antibody. For example, the beads may be magnetic beads such as, e.g.,DYNABEADS, coated with anti-CD3 and anti-CD28. The anti-CD3 andanti-CD28 beads can suitably provide the stimulatory signals to supportT cell activation. See, e.g., Riddell 1990; Trickett 2003.

In other embodiments, the cell culture is activated with a solubleantibody. In further embodiments, the soluble antibody is a solubleanti-CD3 antibody. OKT3 is a murine monoclonal antibody of theimmunoglobulin IgG2a isotype and targets CD3. Thus, in some embodiments,the soluble anti-CD3 antibody is OKT3. OKT3 is further described in,e.g., Dudley 2003; Manger 1985; Ceuppens 1985; Van Wauwe 1980; Norman1995.

In some embodiments, the co-stimulatory signal for T cell activation isprovided by accessory cells. Accessory cells may include, for example, aFc receptor, which enables cross-linking of the CD3 antibody with theTCR/CD3 complex on the T cell. In some embodiments, the cell culture isa mixed population of peripheral blood mononuclear cells (PBMCs). PBMCmay include accessory cells capable of supporting T cell activation. Forexample, CD28 co-stimulatory signals can be provided by the B7 moleculespresent on monocytes in the PBMC. Accordingly, in some embodiments, theaccessory cells include a monocyte or a monocyte-derived cell (e.g., adendritic cell). In additional embodiments, the accessory cells includeB7, CD28, and/or ICOS. Accessory cells are further described in, e.g.,Wolf 1994; Chai 1997; Verwilghen 1991; Schwartz 1990; Ju 2003; Baroja1989; Austyn 1987; Tax 1983.

As described herein, activation reagent may determine the phenotype ofthe CAR T cells produced, allowing for the promotion of a desiredphenotype. In some embodiments, the activation reagent determines theratio of T cell subsets, i.e., CD4+ helper T cells and CD8+ cytotoxic Tcells. The cytotoxic CD8+ T cells are typically responsible for killingcancer cells (i.e., the anti-tumor response), cells that are infected(e.g., with viruses), or cells that are damaged in other ways. CD4+ Tcells typically produce cytokines and help to modulate the immuneresponse, and in some cases may support cell lysis. CD4+ cells activateAPCs, which then primes naïve CD8+ T cells for the anti-tumor response.Accordingly, in embodiments, the methods of the present disclosurefurther include producing CAR T cells of a pre-defined phenotype (i.e.,promoting cells of a desired phenotype). The pre-defined phenotype maybe, for example, a pre-defined ratio of CD8+ cells to CD4+ cells. Insome embodiments, the ratio of CD8+ cells to CD4+ cells in a populationof CAR T cells is about 1:1, about 0.25:1, or about 0.5:1. In otherembodiments, the ratio of CD8+ cells to CD4+ cells in a population ofCAR T cells is about 2:1, about 3:1, about 4:1, or about 5:1.

In embodiments, the activation reagent is removed from the activated Tcell culture after the activation step. The activation reagent, e.g., ananti-CD3 antibody and/or an anti-CD28 antibody may be present in thecell culture media. Thus, in some embodiments, the cell culture mediacontaining the activation reagent, e.g., an anti-CD3 antibody and/or ananti-CD28 antibody, is removed from the activated T cell culture afterthe activation step. In some embodiments, removal of the activationreagent includes removing a soluble antibody. For example, the solubleantibody can be removed by exchanging the cell culture media. Thesoluble antibody can also be removed by affinity methods specific forthe soluble antibody. In other embodiments, removal of the activationreagent includes removing the bead containing the antibody. Bead removalcan include, for example, filtering the beads or removal by a magnet.

Transduction of Activated T Cells. In some embodiments, the geneticallymodified immune cell culture is an activated T cell culture that istransduced with a vector encoding a chimeric antigen receptor to producea transduced T cell culture. In some embodiments, the transductionincludes viral infection, transposons, mRNA transfection,electroporation, or combinations thereof. In some embodiments, thetransduction includes electroporation. Accordingly, in embodiments, thecell engineering system includes an electroporation system orelectroporation unit, as described herein. In additional embodiments,the transduction includes viral infection. The vector may be a viralvector, such as, for example, a lentiviral vector, a gammaretroviralvector, an adeno-associated viral vector, or an adenoviral vector. Inembodiments, the transduction includes introducing a viral vector intothe activated T cells of the cell culture. In additional embodiments,the vector is delivered as a viral particle.

In some embodiments, the transduction step includes transducing theactivated T cells with a lentiviral vector, wherein the lentiviralvector is introduced at a multiplicity of infection (MOI) of about 0.5to about 50, about 0.5 to about 30, or about 0.5 to about 20. In someembodiments, the lentiviral vector is introduced at a MOI of about 0.5to about 8. In some embodiments, the lentiviral vector is introduced ata MOI of about 0.5 to about 6. In some embodiments, the lentiviralvector is introduced at a MOI of about 0.5 to about 4. In someembodiments, the lentiviral vector is introduced at a MOI of about 0.5to about 2. In some embodiments, the lentiviral vector is introduced ata MOI of about 0.6 to about 1.5. In some embodiments, the lentiviralvector is introduced at a MOI of about 0.7 to about 1.3. In someembodiments, the lentiviral vector is introduced at a MOI of about 0.8to about 1.1. In some embodiments, the lentiviral vector is introducedat a MOI of about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6,about 1.7, about 1.8, about 1.9, or about 2.

In some embodiments, after the activation step, the cell culture mediafrom the T cell culture is removed, and the media is then mixed with thevector (e.g., lentiviral vector) and distributed uniformly to the cells.In some embodiments, the removed cell culture media is used to diluteand uniformly deliver the vector to the activated T cell culture.Uniform distribution and consequent homogeneous exposure of the vector(e.g., lentiviral vector) in the T cell culture improves transductionefficiency. In some embodiments, the volume of the cell culture isreduced after activation, and prior to addition of the vector. Volumereduction may enable a higher degree of cell-vector contact. In someembodiments, the activated T cell culture is substantially undisturbedduring the transduction. In some embodiments, the cell culture issubstantially undisturbed during the activation and transduction steps,i.e., the cells remain generally in the same area of the chamber (e.g.,the bottom of the cell culture chamber) while the activation reagent orthe vector is being provided to the cells. This may facilitate uniformdistribution and homogeneous exposure of the activation reagent and/orvector to the cells, and thus may improve the activation and/ortransduction efficiency.

Accordingly, in some embodiments, the transduction efficiency of themethod using the cell engineering system is higher than the transductionefficiency of a method using a flexible, gas-permeable bag for cellculture. In some embodiments, the transduction efficiency of the methodfor automated production of CAR T cells as described herein has at least10% greater, at least 15% greater, at least 20% greater, at least 25%greater, at least 30% greater, at least 35% greater, at least 40%greater, at least 45% greater, at least 50% greater, at least 55%greater, at least 60% greater, at least 65% greater, at least 70%greater, at least 75% greater, at least 80% greater, at least 85%greater, at least 90% greater, at least 95% greater, or at least 100%greater than the transduction efficiency of a method utilizing aflexible, gas-permeable bag.

Expansion of Transduced T Cells. In some embodiments, the transduced Tcell culture (or other immune cell culture) is expanded to a pre-definedculture size (i.e., number of cells). The pre-defined culture size mayinclude a sufficient number of cells suitable for clinical use, i.e.,transfusion into a patient, research and development work, etc. In someembodiments, a clinical or therapeutic dose of CAR T cells foradministration to a patient is about 10⁵ cells, about 10⁶ cells, about10⁷ cells, about 10⁸ cells, about 10⁹ cells, or about 10¹⁰ cells. Insome embodiments, the method produces at least 1, at least 2, at least3, at least 4, at least 5, at least 10, at least 15, at least 20, atleast 25, at least 30, at least 35, at least 40, at least 45, at least50, at least 60, at least 70, at least 80, at least 90, or at least 100clinical doses of CAR T cells. In some embodiments, the transduced Tcell culture is expanded to a total volume of from about 0.1 L to about5 L, from about 0.1 L to about 2 L, or from about 0.2 L to about 2 L. Insome embodiments, the transduced T cell culture is expanded to a totalvolume of about 0.1 L, about 0.2 L, about 0.3 L, about 0.4 L, about 0.5L, about 0.6 L, about 0.7 L, about 0.8 L, about 0.9 L or about 1.0 L.The volume can also be varied through the process, as required based onthe stage of the cell production process. In some embodiments, thepre-defined culture size is input by a user of the cell engineeringsystem. The user may input the pre-defined culture size as a desiredcell count to be produced (e.g., 10¹⁰ CART cells), or, the pre-definedculture size may be input as a desired number of clinical or therapeuticdoses to be produced (e.g., 10 clinical or therapeutic doses of CARTcells). In embodiments, the number of CAR T cells produced by themethods described herein is at least about 100 million (i.e., 1*10 ⁶)cells, or at least about 300 million, at least about 500 million, atleast about 600 million, at least about 700 million, at least about 800million, at least about 900 million, at least about 1 billion (i.e.,1*10 ⁹), at least about 1.1 billion, at least about 1.2 billion, atleast about 1.3 billion, at least about 1.4 billion, at least about 1.5billion, at least about 1.6 billion, at least about 1.7 billion, atleast about 1.8 billion, at least about 1.9 billion, at least about 2billion (i.e., 2*10 ⁹) cells, including at least about 2.1 billion, atleast about 2.2 billion, at least about 2.3 billion, at least about 2.4billion, at least about 2.5 billion, at least about 2.6 billion, atleast about 2.7 billion, at least about 2.8 billion, at least about 2.9billion, or at least about 3.0 billion CAR T cells.

In some embodiments, the expanding of the transduced T cell cultureincludes at least one round of feeding, washing, monitoring, andselecting of the transduced T cell culture. Feeding the cell culture mayinclude supplementing the cell culture with media and/or additionalnutrients. Washing the cell culture may include removing spent media(i.e., media that is depleted of nutrients and/or contains cellularwaste products) and replenishing the cell culture with fresh media.Monitoring the cell culture may include monitoring the temperature, pH,glucose, oxygen level, carbon dioxide level, and/or optical density ofthe cell culture. Selecting the cell culture may include selecting thecells with the desired characteristics such as, e.g., viability, type,and/or morphology, and removing cells that do not have the desiredcharacteristics. In some embodiments, the cell engineering system isconfigured to perform several rounds of the feeding, washing,monitoring, and/or selecting of the transduced T cell culture to achievethe pre-defined culture size. In some embodiments, the cell engineeringsystem performs at least 2, at least 3, at least 4, at least 5, at least6, at least 7, at least 8, at least 9, at least 10, at least 15, atleast 20, at least 25, at least 30, at least 35, at least 40, at least45, at least 50, or at least 100 rounds of the feeding, washing,monitoring, and/or selecting of the transduced T cell culture to achievethe pre-defined culture size.

In embodiments, one or more of the feeding, washing and monitoring canbe removed, or the order of the events can be changed depending on thedesired cellular phenotype or number of cells, etc.

In embodiments, the monitoring includes monitoring with a temperaturesensor, a pH sensor, a glucose sensor, an oxygen sensor, a carbondioxide sensor, and/or an optical density sensor. Accordingly, in someembodiments, the cell engineering system includes one or more of atemperature sensor, a pH sensor, a glucose sensor, an oxygen sensor, acarbon dioxide sensor, and/or an optical density sensor. In additionalembodiments, the cell engineering system is configured to adjust thetemperature, pH, glucose, oxygen level, carbon dioxide level, and/oroptical density of the cell culture, based on the pre-defined culturesize. For example, if the cell engineering system detects that thecurrent oxygen level of the cell culture is too low to achieve thenecessary growth for a desired cell culture size, the cell engineeringsystem will automatically increase the oxygen level of the cell cultureby, e.g., introducing oxygenated cell culture media, by replacing thecell culture media with oxygenated cell culture media, or by flowing thecell culture media through an oxygenation component (i.e., a siliconetubing). In another example, if the cell engineering system detects thatthe current temperature of the cell culture is too high and that thecells are growing too rapidly (e.g., possible overcrowding of the cellsmay lead to undesirable characteristics), the cell engineering systemwill automatically decrease the temperature of the cell culture tomaintain a steady growth rate (or exponential growth rate, as desired)of the cells. In still further embodiments, the cell engineering systemautomatically adjusts the schedule of cell feeding (i.e., providingfresh media and/or nutrients to the cell culture) based on the cellgrowth rate and/or cell count, or other monitored factors, such as pH,oxygen, glucose, etc. The cell engineering system may be configured tostore media (and other reagents, such as wash solutions, etc.) in alow-temperature chamber (e.g., 4° C. or −20° C.), and to warm the mediain a room temperature chamber or a high-temperature chamber (e.g., 25°C. or 37° C., respectively) before introducing the warmed media to thecell culture.

In embodiments, the washing includes washing the cells by filtration orsedimentation. In some embodiments, the washing step does not requiremoving the cell culture vessels or flasks, i.e., the cells can be washedin the same cell culture vessel or flask. In further embodiments, thecells remain substantially undisturbed during the washing step. Inembodiments, the selecting includes mixing the cell culture with one ormore selection reagents. The selection reagent may be a bead, e.g., amagnetic bead, that is specific for the desired cell type, and the cellsbound to the beads are then separated from non-bound cells, e.g., bypassing through a magnetic chamber. For example, the selection beadincludes an antibody specific for a desired cell type, e.g., an anti-CD8antibody or an anti-CD4 antibody. Selection can also be performed byfiltration to remove or select certain cell types based on size. Cellselection by plastic-adhesion (i.e. cells can start in one chamber, theunwanted cells stick to the surface and then the desired cells, that arestill in suspension, are moved to another chamber), can also beutilized.

Suitably, during the expansion stage, the cells are not shaken orrotated. It has been determined that maintaining the cells in arelatively stationary position during expansion helps aid in overallcell production, as well as providing the desired cellular phenotype.

Concentration of the Expanded Culture. In some embodiments, the expandedT cell culture (or other immune cell culture) is concentrated to apre-defined concentration. The pre-defined concentration is of a volumethat can be suitably infused into a patient. For example, the expanded Tcell culture can be concentrated to about 1 ml, about 2 ml, about 5 ml,about 10 ml, about 15 ml, about 20 ml, about 25 ml, about 30 ml, about35 ml, about 40 ml, about 45 ml, about 50 ml, about 55 ml, about 60 ml,about 65 ml, about 70 ml, about 75 ml, about 80 ml, about 85 ml, about90 ml, about 95 ml, or about 100 ml. In some embodiments, theconcentration is performed by centrifugation. In some embodiments, theconcentration is performed by filtration. In some embodiments, thefiltration is ultrafiltration and/or diafiltration. In some embodiments,the pre-defined concentration is input by a user of the cell engineeringsystem. In other embodiments, the pre-defined concentration isdetermined by the cell engineering system, based on a differentparameter input by the user, for example, the number or volume ofclinical or therapeutic doses to be produced; or the number of cells tobe produced. In some embodiments, the cell engineering systemautomatically adjusts the volume or number of clinical or therapeuticdoses produced, based on the input parameters. In some embodiments, thecell engineering system automatically adjusts parameters of thecentrifugation (e.g., speed, duration of centrifuging) or filtration(e.g., filter size, volume, duration) based on the pre-definedconcentration.

Sedimentation based on the port position and design of the chamber canalso be utilized. That is, the fluid volume can be reduced in thechamber to approximately 0.5 mL without removing the cells.

CAR T Cell Culture Harvest. In some embodiments, the concentrated T cellculture (or other immune cell culture) is harvested, suitably to producea chimeric antigen receptor (CAR) T cell culture. In some embodiments,the harvesting includes agitation, fluid flow, and washing of the CAR Tcells. In some embodiments, the harvesting includes separation of thecells from undesired products, which include, e.g., cellular wasteproducts, selection reagents such as beads (e.g., beads containingantibodies and/or beads used for separation of cells), or excess viralvectors. In some embodiments, the harvesting includes uniformdistribution of the CAR T cells into one or more flasks, vials orvessels. In some embodiments, the harvesting includes resuspending theCAR T cells in a formulation reagent, e.g., a solution that stabilizesthe CAR T cells for long-term storage. In some embodiments, theharvesting includes cryopreservation of the CAR T cells.

Further Downstream Processes. In some embodiments, the CAR T cellsundergo further downstream processing prior to therapeutic use in apatient. For example, the cryopreserved CAR T cells may be filtered bysterile filtration to remove potential viral particle remnants. Aftersterile filtration, the CAR T cells may undergo at least one moreconcentration step before packaged in one or more vials, flasks,vessels, or containers. The packaged CAR T cells may be subjected toquality assessment and/or quality control testing. In some embodiments,the CAR T cells undergo minimal downstream processing prior toadministration to a patient. For example, in some embodiments, harvestedCAR T cells are not cryopreserved but transferred to the patient withina short time period after harvest. Avoiding the cryopreservation stepmay increase the viability of the cells.

Cell Engineering Systems. In some embodiments, the methods describedherein are performed by a fully enclosed cell engineering system 600(see FIGS. 6A, 6B), suitably having instructions thereon for performingthe activating, transducing, expanding, concentrating, and harvestingsteps. Cell engineering systems for automated production of geneticallymodified immune cells, including CAR T cells, are described herein, andare also called automated cell engineering system, COCOON, or COCOONsystem throughout. For example, a user can provide a cell engineeringsystem pre-filled with a cell culture and reagents (e.g., an activationreagent, a vector, cell culture media, nutrients, selection reagent, andthe like) and parameters for the cell production (e.g., starting numberof cells, type of media, type of activation reagent, type of vector,number of cells or doses to be produced, and the like), the cellengineering system is able to carry out the methods of producinggenetically modified immune cell cultures, including CAR T cells,without further input from the user. At the end of the automatedproduction process, the cell engineering system may alert the user(e.g., by playing an alert message or sending a mobile app alert) forcollecting the produced cells. In some embodiments, the fully enclosedcell engineering system includes sterile cell culture chambers. In someembodiments, the fully enclosed cell engineering system minimizescontamination of the cell cultures by reducing exposure of the cellculture to non-sterile environments. In additional embodiments, thefully enclosed cell engineering system minimizes contamination of thecell cultures by reducing user handling of the cells.

As described herein, the cell engineering systems suitably include acassette 602. Thus, in embodiments, provided herein is a cassette foruse in an automated cell engineering system. As used herein a “cassette”refers to a largely self-contained, removable and replaceable element ofa cell engineering system that includes one or more chambers forcarrying out the various elements of the methods described herein, andsuitably also includes one or more of a cell media, an activationreagent, a vector, etc.

FIG. 6B shows an embodiments of a cassette 602 in accordance withembodiments hereof. In embodiments, cassette 602 includes a lowtemperature chamber 604, suitably for storage of a cell culture media,as well as a high temperature chamber 606, suitably for carrying outactivation, transduction and/or expansion of an immune cell culture.Suitably, high temperature chamber 606 is separated from low temperaturechamber 606 by a thermal barrier 1102 (see FIG. 11B). As used herein“low temperature chamber” refers to a chamber, suitably maintained belowroom temperature, and more suitably from about 4° C. to about 8° C., formaintenance of cell media, etc., at a refrigerated temperature. The lowtemperature chamber can include a bag or other holder for media,including about 1 L, about 2 L, about 3 L, about 4 L, or about 5 L offluid. Additional media bags or other fluid sources can be connectedexternally to the cassette, and connected to the cassette via an accessport.

As used herein “high temperature chamber” refers to chamber, suitablymaintained above room temperature, and more suitably maintained at atemperature to allow for cell proliferation and growth, i.e., betweenabout 35-40° C., and more suitably about 37° C.

In embodiments, high temperature chamber 606 suitably includes a cellculture chamber 610 (also called proliferation chamber or cellproliferation chamber throughout), as shown in FIG. 6D and FIG. 6E.

The cassettes further include one or more fluidics pathways connected tothe cell culture chamber, wherein the fluidics pathways providerecirculation, removal of waste and homogenous gas exchange anddistribution of nutrients to the cell culture chamber without disturbingcells within the cell culture chamber. Cassette 602 also furtherincludes one or more pumps 605, including peristaltic pumps, for drivingfluid through the cassette, as described herein, as well as one or morevalves 607, for controlling the flow through the various fluidicpathways.

In exemplary embodiments, as shown in FIG. 6D, cell culture chamber 610is flat and non-flexible chamber (i.e., made of a substantiallynon-flexible material such as a plastic) that does not readily bend orflex. The use of a non-flexible chamber allows the cells to bemaintained in a substantially undisturbed state. As shown in FIG. 6E,cell culture chamber 610 is oriented so as to allow the immune cellculture to spread across the bottom 612 of the cell culture chamber. Asshown in FIG. 6E, cell culture chamber 610 is suitably maintained in aposition that is parallel with the floor or table, maintaining the cellculture in an undisturbed state, allowing the cell culture to spreadacross a large area of the bottom 612 of the cell culture chamber. Inembodiments, the overall thickness of cell culture chamber 610 (i.e.,the chamber height 642) is low, on the order of about 0.5 cm to about 5cm. Suitably, the cell culture chamber has a volume of between about0.50 ml and about 300 ml, more suitably between about 50 ml and about200 ml, or the cell culture chamber has a volume of about 180 ml. Theuse of a low chamber height 642 (less than 5 cm, suitably less than 4cm, less than 3 cm, or less then 2 cm) allows for effective media andgas exchange in close proximity to the cells. Ports are configured toallow mixing via recirculation of the fluid without disturbing thecells. Larger height static vessels can produce concentration gradients,causing the area near the cells to be limited in oxygen and freshnutrients. Through controlled flow dynamics, media exchanges can beperformed without cell disturbance. Media can be removed from theadditional chambers (no cells present) without risk of cell loss.

As described herein, in exemplary embodiments the cassette is pre-filledwith one or more of a cell culture, a culture media, an activationreagent, and/or a vector, including any combination of these. In furtherembodiments, these various elements can be added later via suitableinjection ports, etc.

As described herein, in embodiments, the cassettes suitably furtherinclude one or more of a pH sensor, a glucose sensor, an oxygen sensor,a carbon dioxide sensor, a lactic acid sensor/monitor, and/or an opticaldensity sensor. The cassettes can also include one or more samplingports and/or injection ports. Examples of such sampling ports andinjection ports (1104) are illustrated in FIG. 11A, and can include anaccess port for connecting the cartridge to an external device, such asan electroporation unit or an additional media source. FIG. 11A alsoshows the location of the cell input 1105, reagent warming bag 1106which can be used to warm cell media, etc., as well as the culture zone1107, which holds various components for use in the culture media,including for example, cell media, vectors, nutrients and wasteproducts, etc.

FIG. 11B shows the COCOON cell engineering system with cassette 602removed. Visible in FIG. 11B are components of the cell engineeringsystem, including gas control seal 1120, warming zone 1121, actuators1122, pivot 1123 for rocking or tilting the cell engineering system asdesired, and low temperature zone 1124 for holding low temperaturechamber 606. Also shown is an exemplary user interface 1130, which caninclude a bar code reader, and the ability to receive using inputs bytouch pad or other similar device. FIG. 11E shows an additional detailedview of cassette 602, including the location of secondary chamber 1150,which can be used is additional cell culture volume is required, as wellas harvesting chamber 1152, which can be used to recover the final cellculture as produced herein.

In exemplary embodiments, as shown in FIG. 6F, cell culture chamber 610further comprises at least one of: a distal port 620 configured to allowfor the removal of air bubbles from the cell culture chamber and/or as arecirculation port; a medial port 622 configured to function as arecirculation inlet port; and a proximal port 624 configured to functionas a drain port for cell removal.

In still further embodiments, provided herein is cassette 602 for use inan automated cell engineering system 600, comprising cell culturechamber 610 for carrying out activation, transduction and/or expansionof an immune cell culture having a chamber volume that is configured tohouse an immune cell culture and a satellite volume 630 for increasingthe working volume of the cell culture chamber by providing additionalvolume for media and other working fluids without housing the immunecell culture (i.e., satellite volume does not contain any cells).Suitably, the satellite volume is fluidly connected to the cell culturechamber such that media is exchanged with the culture chamber withoutdisturbing the immune cell culture. In exemplary embodiments, satellitevolume is a bag, and in other embodiments, satellite volume is anon-yielding chamber. In embodiments, the satellite volume is betweenabout 0.50 ml and about 300 ml, more suitably between about 150 ml andabout 200 ml. FIG. 6D-6E show the position of a satellite volume 630 incassette 602.

FIG. 6G shows a schematic illustrating the connection between cellculture chamber 610, and satellite volume 630. Also illustrated in FIG.6G are the positioning of various sensors (e.g., pH sensor 650,dissolved oxygen sensor 651), as well as sampling/sample ports 652 andvarious valves (control valves 653, bypass check valves 654), as well asone or more fluidic pathways 640, suitably comprising a silicone-basedtubing component, connecting the components. As described herein, use ofa silicone-based tubing component allows oxygenation through the tubingcomponent to facilitate gas transfer and optimal oxygenation for thecell culture. Also show in FIG. 6G is the use of one or more hydrophobicfilters 655 or hydrophilic filters 656, in the flow path of thecassette, along with pump tube 657 and bag/valve module 658.

FIG. 6H shows gas exchange data using the COCOON system, as compared totraditional bags.

In embodiments, satellite volume 630 is further configured to allowmedia removal without loss of cells of the immune cell culture. That is,the media exchange between the satellite volume and the cell culturechamber is performed in such a manner that the cells are not disturbedand are not removed from the cell culture chamber.

In additional embodiments, as shown in FIG. 6G, cassette 602 suitablyfurther includes a crossflow reservoir 632 for holding additional media,etc., as needed. Suitably, the crossflow reservoir has a volume ofbetween about 0.50 ml and about 300 ml, more suitably between about 100ml and about 150 ml.

The cell engineering systems described herein suitably have threerelevant volumes, the cell culture chamber volume, the working volume,and the total volume. Suitably, the working volume used in the cassetteranges from 180 mL to 460 mL based on the process step, and can beincreased up to about 500 mL, about 600 mL, about 700 mL, about 800 mL,about 900 mL or about 1 L. In embodiments, the cassette can readilyachieve 4*10⁹ cells-10*10⁹ cells. The cell concentration during theprocess varies from 0.3*10⁶ cells/ml to approximately 10*10⁶ cells/ml.The cells are located in the cell culture chamber, but media iscontinuously recirculated through additional chambers (e.g., crossflowreservoir and satellite volume) to increase the working volume, asdescribed herein.

As described herein, unlike a flexible bag, which changes shape whenfilled with liquid (e.g., a cell culture) and when picked up or moved, a“substantially non-yielding chamber” (e.g., an exemplary cell culturechamber 610) does not change shape (e.g., bend, curve, or deform) whenfilled with liquid, picked up, or moved during typical handlingconditions. Thus, in some embodiments, a substantially non-yieldingchamber allows cells to remain substantially in the same area of thechamber, even when the chamber is picked up or moved. A substantiallynon-yielding chamber also does not have the curvature associated with abag. Thus, in some embodiments, the cells are distributed more uniformlyin a substantially non-yielding chamber compared with a bag. In someembodiments, the activation reagent and/or the vector are distributedmore uniformly in a substantially non-yielding chamber compared with abag.

In some embodiments, the cell engineering system includes a plurality ofchambers. In further embodiments, each of the activating, transducing,expanding, concentrating, and harvesting steps of the method for cellsdescribed herein is performed in a different chamber of the plurality ofchambers of the cell engineering system. In some embodiments, the cellsare substantially undisturbed during transfer from one chamber toanother. In other embodiments, the steps of the method are performed inthe same chamber of the cell engineering system, and the cellengineering system automatically adjusts the chamber environment asneeded for each step of the method. Thus further allows for the cells tonot be disturbed during the various steps.

In some embodiments, the cell engineering system has improved gasexchange compared with a flexible, gas-permeable bag for cell culture.In some embodiments, the cell engineering system includes gas exchangelines. The gas exchange lines may be made from a gas-permeable materialsuch as, e.g., silicone. In some embodiments, the gas permeabilitycoefficient of the gas exchange lines is higher than the permeabilitycoefficient of the material used in the flexible, gas-permeable bag. Insome embodiments, the cell engineering system recirculates oxygenthroughout the substantially non-yielding chamber during the cellproduction methods. Thus, in some embodiments, the oxygen level of acell culture in the cell engineering system is higher than the oxygenlevel of a cell culture in a flexible, gas-permeable bag. Higher oxygenlevels may be important in the cell culture expansion step, as increasedoxygen levels may support increased cell growth and proliferation.

In some embodiments, the cell engineering system continuouslyrecirculates media throughout the chambers without disturbing the cells.For example, the cell engineering system can continuously replenishnutrients, remove waste, and circulate released cytokines and dissolvedgases through the chamber, while the cells remain in the same area ofthe chamber. The continuous circulation can improve the uniformdistribution of positive factors and uniform removal of negativefactors, which reduces localized effects that are caused by unevendistribution, without disturbing the cells.

In some embodiments, the cell engineering system provides carbon dioxidethroughout the chamber during the cell production methods (including CART production). CO₂ can help to maintain a target pH in the cell culture,which can be important for cell growth and proliferation. In someembodiments, the cell engineering system monitors the CO₂ level of thecell culture and adjusts the amount of CO₂ provided based on themeasured CO₂ level. For example, as the cell culture increases, there isa corresponding increase in the amount of CO₂ produced by the cells, andthe cell engineering system reduces the amount of CO₂ provided. Thedesired CO₂ level of the cell culture may be defined by the user, forexample, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, or about 10% CO₂. Since the cellengineering system is constantly adjusting the amount of CO₂ providedbased on the measured CO₂ level of the cell culture, the cellengineering system is able to maintain a desired CO₂ level throughoutthe production process. The amount of CO₂ in a cell culture may alsoaffect the pH of the culture, since dissolved CO₂ generally acidifies asolution (through reacting with water to form carbonic acid). Thus,maintaining a steady CO₂ level in the cell culture may result in a morestable pH. Accordingly, in embodiments, the pH level of the cell cultureremains substantially constant during the production process. In furtherembodiments, the pH level of the transduced cell culture remainssubstantially constant during the expansion step.

Yields from genetically modified immune cell production, including CAR Tcell production, may be influenced by activation and transductionefficiency, as well as growth conditions of the cells. Activationefficiency can improve with more stable contact between the cells andthe activation reagent. Movement of the cells throughout the culturevessel may lead to an uneven distribution of the cells, and thus createlocalized effects when activation reagent is added to the cell culturechamber. In contrast to a flexible culture bag, cells grown in anon-yielding chamber remain undisturbed during the activation process,which may contribute to a higher activation efficiency.

Improving activation efficiency may also lead to greater vectortransduction efficiency. If cells are activated and are activelydividing, the vector (e.g., lentiviral vector) could integrate moreeffectively into the cells. Homogeneous distribution of the cells in thecell culture chamber 610 may facilitate homogeneous exposure of thevector to the cells, whereas cells may be unevenly distributed, and thusreceive different vector exposure, in a flexible cell culture bag. Thus,in some embodiments, the transduction efficiency of the method forautomated production of genetically modified immune cells, includingCART cells as described herein, is at least 10% greater, at least 15%greater, at least 20% greater, at least 25% greater, at least 30%greater, at least 35% greater, at least 40% greater, at least 45%greater, at least 50% greater, at least 55% greater, at least 60%greater, at least 65% greater, at least 70% greater, at least 75%greater, at least 80% greater, at least 85% greater, at least 90%greater, at least 95% greater, or at least 100% greater than thetransduction efficiency of a method utilizing a flexible, gas-permeablebag.

Growth conditions of the cell cultures may also improve cell yields. Forexample, higher oxygen levels in the cell engineering system,facilitated by highly gas-permeable tubing and continuous recirculationof oxygen in the cell culture chamber, may increase cell proliferation.The ability of the cell engineering system to constantly monitor thestate of the cell culture, and make adjustments accordingly, may also beadvantageous. For example, the cell engineering system can monitor theCO₂ O₂, N₂, and/or pH level of the cell culture and adjust the level ofCO₂ O₂, or N₂. Nutrients can also be provided in a timely and consistentmanner and distributed uniformly to the cell culture. Thus, theautomated methods for producing genetically modified immune cells,including CAR T cells, described herein advantageously results in highercell yields compared with manual methods, or methods utilizing aflexible culture bag. Accordingly, in some embodiments, the method forautomated production of genetically modified immune cells, including CART cells utilizing a cell engineering system as described herein,produces at least 10% more, at least 15% more at least 20% more, atleast 25% more at least 30% more, at least 35% more at least 40% more,at least 45% more at least 50% more, at least 55% more at least 60%more, at least 65% more at least 70% more, at least 75% more at least80% more, at least 85% more at least 90% more, at least 95% more or atleast 100% more cells than a method utilizing a flexible, gas permeablebag for cell culture. In embodiments, the number of cells produced bythe methods described herein is at least about 2 billion (i.e., 2*10⁹)cells, including at least about 2.1 billion, at least about 2.2 billion,at least about 2.3 billion, at least about 2.4 billion, at least about2.5 billion, at least about 2.6 billion, at least about 2.7 billion, atleast about 2.8 billion, at least about 2.9 billion, or at least about3.0 billion cells.

Additional Exemplary Embodiments

Embodiment 1 is a method for automated production of a geneticallymodified immune cell culture, the method comprising activating an immunecell culture with an activation reagent to produce an activated immunecell culture, transducing the activated immune cell culture with avector, to produce a transduced immune cell culture, expanding thetransduced immune cell culture, concentrating the expanded immune cellculture, and harvesting the concentrated immune cell culture to producea genetically modified immune cell culture, further comprising washingeither or both the expanded immune cell culture and the concentratedimmune cell culture, wherein the steps are performed by a fully enclosedcell engineering system and the steps are optimized via a process toproduce the genetically modified immune cell culture.

Embodiment 2 includes the method of embodiment 1, wherein the process isa self-adjusting process and includes monitoring with one or more of atemperature sensor, a pH sensor, a glucose sensor, an oxygen sensor, acarbon dioxide sensor, and an optical density sensor; and adjusting oneor more of a temperature, a pH level, a glucose level, an oxygen level,a carbon dioxide level, and an optical density of the transduced T cellculture, based on the monitoring.

Embodiment 3 includes the method of embodiments 1-2, wherein the methodproduces at least about 100 million viable genetically modified immunecells

Embodiment 4 includes the method of embodiments 1-3, wherein the methodproduces at least about 2 billion viable genetically modified immunecells

Embodiment 5 includes the method of embodiments 1-4, wherein the immunecell culture is a T cell culture.

Embodiment 6 includes the method of embodiment 5, wherein T cell cultureis a chimeric antigen receptor T (CAR T) cell culture.

Embodiment 7 includes the method of embodiment 6, wherein the vectorencodes a chimeric antigen receptor.

Embodiment 8 includes the method of embodiments 1-7, wherein the immunecell culture comprises peripheral blood mononuclear cells and/orpurified T cells.

Embodiment 9 includes the method of embodiments 1-8, wherein the immunecell culture comprises at least one accessory cell.

Embodiment 10 includes the method of embodiment 9, wherein the accessorycell comprises a monocyte or a monocyte-derived cell.

Embodiment 11 includes the method of embodiment 9, wherein the accessorycell comprises antigens for a T cell receptor, including CD28, CD40,CD2, CD40L and/or ICOS.

Embodiment 12 includes the method of embodiments 1 to 11, wherein theactivation reagent comprises an antibody or a dendritic cell.

Embodiment 13 includes the method of embodiment 12, wherein the antibodyis immobilized on a surface.

Embodiment 14 includes the method of embodiment 13, wherein the surfaceis a surface of a bead.

Embodiment 15 includes the method of embodiment 12, wherein the antibodyis a soluble antibody.

Embodiment 16 includes the method of embodiments 12-15, wherein theantibody comprises at least one of an anti-CD3 antibody and an anti-CD28antibody.

Embodiment 17 includes the method of embodiments 1-16, wherein thetransducing comprises viral infection, electroporation, membranedisruption, or combinations thereof.

Embodiment 18 includes the method of embodiments 1-17, wherein thevector is a lentiviral vector or a retrovirus.

Embodiment 19 includes the method of embodiments 1-18, wherein thetransducing comprises mixing the vector in cell culture media anddelivering the vector in the media uniformly to the activated immunecell culture.

Embodiment 20 includes the method of embodiments 1-19, wherein theexpanding comprises at least one or more of feeding, washing andmonitoring of the transduced immune cell culture.

Embodiment 21 includes the method of embodiments 2-20, wherein theoxygen level of the transduced immune cell culture is optimized for theimmune cell culture.

Embodiment 22 includes the method of embodiments 1-21, wherein the cellengineering system recirculates cell culture media through anoxygenation component during one or more of steps (a) to (e).

Embodiment 23 includes the method of embodiments 1-22, wherein the cellengineering system recirculates nutrients, waste, released cytokines,and/or dissolved gasses during steps (a) to (e).

Embodiment 24 includes the method of embodiments 2-23, wherein thecarbon dioxide level provided by the cell engineering system decreasesduring step (c).

Embodiment 25 includes the method of embodiments 1-24, wherein the cellengineering system is configured to perform several rounds of one ormore of feeding, washing, monitoring, and selecting of the transducedimmune cell culture.

Embodiment 26 includes the method of embodiments 1-25, wherein theconcentrating comprises centrifugation, supernatant removal followingsedimentation, or filtration.

Embodiment 27 includes the method of embodiment 26, wherein the processfurther includes adjusting parameters of the centrifugation orfiltration.

Embodiment 28 includes the method of embodiments 1 to 27, wherein thecell engineering system comprises a plurality of chambers, and whereineach of steps (a) to (e) is performed in a different chamber of theplurality of chambers of the cell engineering system.

Embodiment 29 includes the method of embodiments 1-28, furthercomprising removing the activation reagent from the activated immunecell culture after step (a).

Embodiment 30 includes the method of embodiments 1-29, wherein the cellengineering system contains the cell culture of (a), the activationreagent, the vector, and cell culture medium prior to starting themethod.

Embodiment 31 is a method for promoting a preferred phenotype of agenetically modified immune cell culture, the method comprisingactivating an immune cell culture with an activation reagent to producean activated immune cell culture, wherein the activation reagent andactivating conditions promote the phenotype of the genetically modifiedimmune cell culture, transducing the activated immune cell culture witha vector, to produce a transduced immune cell culture, expanding thetransduced immune cell culture, concentrating the expanded immune cellculture; and harvesting the concentrated immune cell culture to producea genetically modified immune cell culture, wherein the steps areperformed by a fully enclosed, automated cell engineering system.

Embodiment 32 includes the method of embodiment 31, wherein theactivation reagent comprises an antibody or a dendritic cell.

Embodiment 33 includes the method of embodiment 32, wherein the antibodyis immobilized on a surface.

Embodiment 34 includes the method of embodiments 33, wherein the surfaceis a surface of a bead.

Embodiment 35 includes the method of embodiments 32, wherein theantibody is a soluble antibody.

Embodiment 36 includes the method of embodiments 32-35, wherein theantibody comprises at least one of an anti-CD3 antibody, an anti-CD28antibody and an anti-CD2 antibody.

Embodiment 37 includes the method of embodiment 36, wherein the solubleantibody is OKT3.

Embodiment 38 includes the method of embodiments 31-37, wherein theactivating conditions provide a substantially undisturbed immune cellculture allowing for stable contact between the activation reagent andthe immune cell culture.

Embodiment 39 includes the method of embodiments 31-38, wherein themethod produces at least about 100 million viable genetically modifiedimmune cells

Embodiment 40 includes the method of embodiment 39, wherein the methodproduces at least about 2 billion viable genetically modified immunecells

Embodiment 41 includes the method of embodiments 31-40, wherein theimmune cell culture is a T cell culture.

Embodiment 42 includes the method of embodiments 41, wherein T cellculture is a chimeric antigen receptor T (CAR T) cell culture.

Embodiment 43 includes the method of embodiments 42, wherein the vectorencodes a chimeric antigen receptor.

Embodiment 44 includes the method of embodiments 31-43, wherein theimmune cell culture comprises peripheral blood mononuclear cells and/orpurified T cells.

Embodiment 45 includes the method of embodiments 31-44, wherein the cellculture comprises at least one accessory cell.

Embodiment 46 includes the method of embodiment 45, wherein theaccessory cell comprises a monocyte or a monocyte-derived cell.

Embodiment 47 includes the method of embodiment 45, wherein theaccessory cell comprises antigens for a T cell receptor, including CD28,CD40, CD2, CD40L and/or ICOS.

Embodiment 48 includes the method of embodiments 41-47, wherein thephenotype of the T cell culture has a ratio of CD8+ cells to CD4+ ofabout 0.1:1 to about 10:1.

Embodiment 49 includes the method of embodiments 31-48, wherein thetransducing comprises viral infection, electroporation, membranedisruption, or combinations thereof.

Embodiment 50 includes the method of embodiments 31-49, wherein thevector is a lentiviral vector or a retrovirus.

Embodiment 51 includes the method of embodiments 31-50, wherein thetransducing comprises mixing the vector in cell culture media anddelivering the vector in the media uniformly to the activated immunecell culture.

Embodiment 52 includes the method of embodiments 31-51, wherein theexpanding comprises at least one or more of feeding, washing andmonitoring the transduced immune cell culture.

Embodiment 53 includes the method of embodiments 31-52, wherein anoxygen level of the transduced immune cell culture is optimized for thepromoted phenotype.

Embodiment 54 includes the method of embodiments 31-53, wherein the cellengineering system recirculates cell culture media through anoxygenation component during one or more of steps (a) to (e).

Embodiment 55 includes the method of embodiments 31-54, wherein the cellengineering system recirculates nutrients, waste, released cytokines,and/or dissolved gasses during steps (a) to (e).

Embodiment 56 includes the method of embodiments 31-55, wherein a carbondioxide level provided by the cell engineering system decreases duringstep (c).

Embodiment 57 includes the method of embodiments 31-56, wherein the cellengineering system is configured to perform several rounds of thefeeding, washing, monitoring, and selecting of the transduced immunecell culture.

Embodiment 58 includes the method of embodiments 31-57, wherein theconcentrating comprises centrifugation, supernatant removal followingsedimentation, or filtration.

Embodiment 59 includes the method of embodiments 31-58, wherein the cellengineering system comprises a plurality of chambers, and wherein eachof steps (a) to (e) is performed in a different chamber of the pluralityof chambers of the cell engineering system.

Embodiment 60 includes the method of embodiments 31-59, furthercomprising removing the activation reagent from the activated immunecell culture after step (a).

Embodiment 61 includes the method of embodiments 31-60, furthercomprising removing the vector following the transducing in (b).

Embodiment 62 includes the method of embodiments 31-61, wherein the cellengineering system contains the cell culture of (a), the activationreagent, the vector, and cell culture medium prior to starting themethod.

Embodiment 63 is a method for automated production of a geneticallymodified immune cell culture, the method comprising activating an immunecell culture with an activation reagent to produce an activated immunecell culture, transducing the activated immune cell culture with avector, to produce a transduced immune cell culture, expanding thetransduced immune cell culture, concentrating the expanded immune cellculture, and harvesting the concentrated immune cell culture to producea genetically modified immune cell culture, wherein the steps areperformed by a fully enclosed, automated cell engineering system, andwherein each of the steps are performed with immune cell cultures havingan optimized cell density (cells/mL) and an optimized cell confluency(cells/cm²).

Embodiment 64 includes the method of embodiment 63, wherein theoptimized cell density for (a) is about 0.05*106 cells/mL to about60*10⁶ cells/mL.

Embodiment 65 includes the method of embodiments 63 or claim 64, whereinthe optimized cell confluency for (a) is about 0.1*10⁶ cells/cm² toabout 60*10⁶ cells/cm².

Embodiment 66 includes the method of embodiments 63-65, wherein theactivation reagent comprises an antibody or a dendritic cell.

Embodiment 67 includes the method of embodiment 66, wherein the antibodyis immobilized on a surface.

Embodiment 68 includes the method of embodiment 67, wherein the surfaceis a surface of a bead.

Embodiment 69 includes the method of embodiment 66, wherein the antibodyis a soluble antibody.

Embodiment 70 includes the method of embodiments 66-69, wherein theantibody comprises at least one of an anti-CD3 antibody and an anti-CD28antibody.

Embodiment 71 includes the method of embodiments 63-70, wherein themethod produces at least about 100 million viable genetically modifiedimmune cells.

Embodiment 72 includes the method of embodiments 63-71, wherein themethod produces at least about 2 billion viable genetically modifiedimmune cells.

Embodiment 73 includes the method of embodiments 63-72, wherein theimmune cell culture is a T cell culture.

Embodiment 74 includes the method of embodiments 73, wherein T cellculture is a chimeric antigen receptor T (CAR T) cell culture.

Embodiment 75 includes the method of embodiments 74, wherein the vectorencodes a chimeric antigen receptor.

Embodiment 76 includes the method of embodiments 64-75, wherein theimmune cell culture comprises peripheral blood mononuclear cells and/orpurified T cells.

Embodiment 77 includes the method of embodiments 64-76, wherein the cellculture comprises at least one accessory cell.

Embodiment 78 includes the method of embodiment 77 wherein the accessorycell comprises a monocyte.

Embodiment 79 includes the method of embodiment 77, wherein theaccessory cell comprises antigens for a T cell receptor, including CD28,CD40, CD2, CD40L and/or ICOS.

Embodiment 80 includes the method of embodiments 63-79, wherein thetransducing comprises viral infection, electroporation, membranedisruption, or combinations thereof.

Embodiment 81 includes the method of embodiments 63-80, wherein thevector is a lentiviral vector or a retrovirus.

Embodiment 82 includes the method of embodiments 63-81, wherein thetransducing comprises mixing the vector in cell culture media anddelivering the vector in the media uniformly to the activated immunecell culture.

Embodiment 83 includes the method of embodiments 63-82, wherein theexpanding comprises at least one or more of feeding, washing,monitoring, and selecting of the transduced immune cell culture.

Embodiment 84 includes the method of embodiments 63-83, wherein anoxygen level of the transduced immune cell culture is optimized for thecell density and cell confluency.

Embodiment 85 includes the method of embodiments 63-84, wherein the cellengineering system recirculates cell culture media through anoxygenation component during one or more of steps (a) to (e).

Embodiment 86 includes the method of embodiment 85, wherein the oxygenrecirculation is provided by silicone tubing during steps (a) through(c).

Embodiment 87 includes the method of embodiments 63-86, wherein the cellengineering system recirculates nutrients, waste, released cytokines,and/or dissolved gasses during steps (a) to (e).

Embodiment 88 includes the method of embodiments 63-87, wherein a carbondioxide level provided by the cell engineering system decreases duringstep (c).

Embodiment 89 includes the method of embodiments 63-88, wherein therecirculation of nutrients, waste, released cytokines, and/or dissolvedgasses is homogenously provided with the cells having a density of about0.05*10⁶ cells/mL to about 60*10⁶ cells/mL and a confluency of about0.1*10⁶ cells/cm² to about 60*10⁶ cells/cm².

Embodiment 90 includes the method of embodiments 63-89, wherein the cellengineering system is configured to perform several rounds of feeding,washing, monitoring, and selecting of the transduced immune cellculture.

Embodiment 91 includes the method of embodiments 63-90, wherein theconcentrating comprises centrifugation, supernatant removal followingsedimentation, or filtration.

Embodiment 92 includes the method of embodiments 63-91, wherein the cellengineering system comprises a plurality of chambers, and wherein eachof steps (a) to (e) is performed in a different chamber of the pluralityof chambers of the cell engineering system.

Embodiment 93 includes the method of embodiments 63-92, furthercomprising removing the activation reagent from the activated immunecell culture after step (a).

Embodiment 94 includes the method of embodiments 63-93, furthercomprising removing the vector following the transducing in (b).

Embodiment 95 includes the method of embodiments 63-94, wherein the cellengineering system contains the cell culture of (a), the activationreagent, the vector, and cell culture medium prior to starting themethod.

Embodiment 96 is a method for automated production of a geneticallymodified immune cell culture, the method comprising activating an immunecell culture with an activation reagent to produce an activated immunecell culture, transducing the activated immune cell culture with avector, to produce a transduced immune cell culture, expanding thetransduced immune cell culture, wherein the transduced cell culture isnot shaken during the expanding, concentrating the expanded immune cellculture, and harvesting the concentrated immune cell culture to producea genetically modified immune cell culture, wherein the steps areperformed by a fully enclosed, automated cell engineering system.

Embodiment 97 includes the method of embodiment 96, wherein theactivation reagent comprises an antibody or a dendritic cell.

Embodiment 98 includes the method of embodiment 97, wherein the antibodyis immobilized on a surface.

Embodiment 99 includes the method of embodiment 98, wherein the surfaceis a surface of a bead.

Embodiment 100 includes the method of embodiment 97, wherein theantibody is a soluble antibody.

Embodiment 101 includes the method of embodiments 96-100, wherein theantibody comprises at least one of an anti-CD3 antibody, an anti-CD28antibody and an anti-CD2 antibody.

Embodiment 102 includes the method of embodiments 96-101, wherein themethod produces at least about 100 million viable genetically modifiedimmune cells

Embodiment 103 includes the method of embodiment 102, wherein the methodproduces at least about 2 billion viable genetically modified immunecells

Embodiment 104 includes the method of embodiments 96-103, wherein theimmune cell culture is a T cell culture.

Embodiment 105 includes the method of embodiment 104, wherein T cellculture is a chimeric antigen receptor T (CAR T) cell culture.

Embodiment 106 includes the method of embodiment 105, wherein the vectorencodes a chimeric antigen receptor.

Embodiment 107 includes the method of embodiments 96-106, wherein theimmune cell culture comprises peripheral blood mononuclear cells and/orpurified T cells.

Embodiment 108 includes the method of embodiments 96-107, wherein thecell culture comprises at least one accessory cell.

Embodiment 109 includes the method of embodiment 108 wherein theaccessory cell comprises a monocyte or a monocyte-derived cell.

Embodiment 110 includes the method of embodiment 109, wherein theaccessory cell comprises antigens for a T cell receptor, including CD28,CD40, CD2, CD40L and/or ICOS.

Embodiment 111 includes the method of embodiments 96-110, wherein thetransducing comprises viral infection, electroporation, membranedisruption, or combinations thereof.

Embodiment 112 includes the method of embodiments 96-111, wherein thevector is a lentiviral vector or a retrovirus.

Embodiment 113 includes the method of embodiments 96-112, wherein thetransducing comprises mixing the vector in cell culture media anddelivering the vector in the media uniformly to the activated immunecell culture.

Embodiment 114 includes the method of embodiments 96-113, wherein theexpanding comprises at least one or more of feeding, washing,monitoring, and selecting of the transduced immune cell culture, withoutshaking the immune cell culture.

Embodiment 115 includes the method of embodiments 96-114, wherein anoxygen level of the transduced immune cell culture is optimized for theimmune cell culture.

Embodiment 116 includes the method of embodiments 96-115, wherein thecell engineering system recirculates cell culture media through anoxygenation component during one or more of steps (a) to (e).

Embodiment 117 includes the method of embodiments 96-116, wherein thecell engineering system recirculates nutrients, waste, releasedcytokines, and/or dissolved gasses.

Embodiment 118 includes the method of embodiments 96-117, wherein acarbon dioxide level provided by the cell engineering system decreasesduring step (c).

Embodiment 119 includes the method of embodiments 96-118, wherein thecell engineering system is configured to perform several rounds offeeding, washing, monitoring, and selecting of the transduced immunecell culture.

Embodiment 120 includes the method of embodiments 96-119, wherein theconcentrating comprises centrifugation, supernatant removal followingsedimentation, or filtration.

Embodiment 121 includes the method of embodiments 96-120, wherein thecell engineering system comprises a plurality of chambers, and whereineach of steps (a) to (e) is performed in a different chamber of theplurality of chambers of the cell engineering system.

Embodiment 122 includes the method of embodiments 96-121, furthercomprising removing the activation reagent from the activated immunecell culture after step (a).

Embodiment 123 includes the method of embodiments 96-122, furthercomprising removing the vector following the transducing in (b).

Embodiment 124 includes the method of embodiments 96-123, wherein thecell engineering system contains the cell culture of (a), the activationreagent, the vector, and cell culture medium prior to starting themethod.

Embodiment 125 is a method for automated production of a geneticallymodified immune cell culture, the method performed by a cell engineeringsystem, comprising, activating an immune cell culture with an activationreagent to produce an activated immune cell culture in a first chamberof the cell engineering system, transducing the activated immune cellculture, the transducing comprising, transferring the activated immunecell culture from the first chamber to an electroporation unit,electroporating the activated immune cell culture with a vector, toproduce a transduced immune cell culture, transferring the transducedimmune cell culture to a second chamber of the cell engineering system,expanding the transduced immune cell culture, concentrating the expandedimmune cell culture; and harvesting the concentrated immune cell cultureto produce a genetically modified cell culture.

Embodiment 126 includes the method of embodiment 125, wherein thetransducing comprises transferring via a first sterile, closedconnection, the activated immune cell culture from the first chamber tothe electroporation unit, electroporating the activated immune cellculture with the vector, to produce the transduced immune cell culture,transferring via a second sterile, closed connection, the transducedimmune cell culture to the second chamber of the cell engineeringsystem.

Embodiment 127 includes the method of embodiment 126, wherein theelectroporation unit is located outside of the cell engineering system.

Embodiment 128 includes the method of embodiments 125-127, wherein themethod produces at least about 100 million viable genetically modifiedimmune cells.

Embodiment 129 includes the method of embodiment 128, wherein the methodproduces at least about 2 billion viable genetically modified immunecells.

Embodiment 130 includes the method of embodiments 125-129, wherein theimmune cell culture is a T cell culture.

Embodiment 131 includes the method of embodiment 130, wherein T cellculture is a chimeric antigen receptor T (CAR T) cell culture.

Embodiment 132 includes the method of embodiment 131, wherein the vectorencodes a chimeric antigen receptor.

Embodiment 133 includes the method of embodiments 125-132, wherein theimmune cell culture comprises peripheral blood mononuclear cells and/orpurified T cells.

Embodiment 134 includes the method of embodiments 125-132, wherein thecell culture comprises at least one accessory cell.

Embodiment 135 includes the method of embodiment 134, wherein theaccessory cell comprises a monocyte or a monocyte-derived cell.

Embodiment 136 includes the method of embodiments 134, wherein theaccessory cell comprises antigens for a T cell receptor, including CD28,CD40, CD40L and/or ICOS.

Embodiment 137 includes the method of embodiments 125-136, wherein theactivation reagent comprises an antibody or a dendritic cell.

Embodiment 138 includes the method of embodiments 137, wherein theantibody is immobilized on a surface.

Embodiment 139 includes the method of embodiments 138, wherein thesurface is a surface of a bead.

Embodiment 140 includes the method of embodiments 137, wherein theantibody is a soluble antibody.

Embodiment 141 includes the method of embodiments 138-140, wherein theantibody comprises at least one of an anti-CD3 antibody, an anti-CD28antibody and an anti-CD2 antibody.

Embodiment 142 includes the method of embodiments 125-141, wherein thevector is a lentiviral vector or a retrovirus.

Embodiment 143 includes the method of embodiments 125-142, wherein theexpanding comprises at least one or more of feeding, washing,monitoring, and selecting of the transduced immune cell culture.

Embodiment 144 includes the method of embodiments 125-143, wherein anoxygen level of the transduced immune cell culture is optimized for theimmune cell culture.

Embodiment 145 includes the method of embodiments 125-144, wherein thecell engineering system recirculates cell culture media through anoxygenation component during one or more of steps (a) to (e).

Embodiment 146 includes the method of embodiments 125-145, wherein thecell engineering system recirculates nutrients, waste, releasedcytokines, and/or dissolved gasses during steps (a) to (e).

Embodiment 147 includes the method of embodiments 125-146, wherein acarbon dioxide level provided by the cell engineering system decreasesduring step (c).

Embodiment 148 includes the method of embodiments 125-147, wherein thecell engineering system is configured to perform several rounds offeeding, washing, monitoring, and selecting of the transduced immunecell culture.

Embodiment 149 includes the method of embodiments 125-148, wherein theconcentrating comprises centrifugation, supernatant removal followingsedimentation, or filtration.

Embodiment 150 includes the method of embodiments 125-149, wherein thecell engineering system comprises a plurality of chambers, and whereineach of steps (a) to (e) is performed in a different chamber of theplurality of chambers of the cell engineering system.

Embodiment 151 includes the method of embodiments 125-150, furthercomprising removing the activation reagent from the activated immunecell culture after step (a).

Embodiment 152 includes the method of embodiments 125-151, furthercomprising removing the vector following the transducing in (b).

Embodiment 153 includes the method of embodiments 125-152, wherein thecell engineering system contains the cell culture of (a), the activationreagent, the vector, and cell culture medium prior to starting themethod.

Embodiment 154 includes the method of embodiments 1 to 153, whereintransduction efficiency in step (c) of the method is at least 20% higherthan the transduction efficiency of the method utilizing a flexible, gaspermeable bag for cell culture.

Embodiment 155 includes the method of embodiments 1 to 154, wherein themethod produces at least 20% more genetically modified immune cells thana method utilizing manual cell culture with a flexible, gas permeablebag.

Embodiment 156 includes the method of embodiments 1 to 155, wherein thecell engineering system comprises a plurality of chambers, and whereineach of steps (a) to (e) is performed in a different chamber of theplurality of chambers of the cell engineering system, each of (a), theactivation reagent, the vector, and cell culture medium are contained ina different chamber of the plurality of the chambers prior to startingthe method, and wherein at least one of the plurality of chambers ismaintained at a temperature for growing cells and at least one of theplurality of chambers is maintained at a refrigerated temperature.

Embodiment 157 is a cassette for use in an automated cell engineeringsystem, comprising a low temperature chamber, for storage of a cellculture media, a high temperature chamber for carrying out activation,transduction and expansion of an immune cell culture, wherein the hightemperature chamber is separated from the low temperature chamber, by athermal barrier, the high temperature chamber including a cell culturechamber; and one or more fluidics pathways connected to the cell culturechamber, wherein the fluidics pathways provide recirculation, removal ofwaste and homogenous gas exchange and distribution of nutrients to thecell culture chamber without disturbing cells within the cell culturechamber.

Embodiment 158 includes the cassette of embodiment 157, wherein the cellculture chamber is flat and non-flexible chamber, having a low chamberheight.

Embodiment 159 includes the cassette of embodiments 157 or 158, whereinthe cell culture chamber is oriented so as to allow the immune cellculture to spread across the bottom of the cell culture chamber.

Embodiment 160 includes the cassette of embodiments 157-159, wherein thecassette is pre-filled with cell culture, culture media, activationreagent, and a vector.

Embodiment 161 includes the cassette of embodiments 157-160, furthercomprising one or more of a pH sensor, a glucose sensor, an oxygensensor, a carbon dioxide sensor, and/or an optical density sensor.

Embodiment 162 includes the cassette of embodiments 157-161, furthercomprising one or more sampling ports and/or injection ports.

Embodiment 163 includes the cassette of embodiments 157-162, wherein thecell culture chamber further comprises at least one of a distal portconfigured to allow for the removal of air bubbles from the cell culturechamber and/or as a recirculation port; a medial port configured tofunction as a recirculation inlet port; and a proximal port configuredto function as a drain port for cell removal.

Embodiment 164 includes the cassette of embodiments 157-163, furthercomprising an access port for connecting the cartridge to an externaldevice.

Embodiment 165 includes the cassette of embodiment 164, wherein theexternal device includes an electroporation unit or an additional mediasource.

Embodiment 166 is cassette for use in an automated cell engineeringsystem, comprising a cell culture chamber for carrying out activation,transduction and/or expansion of an immune cell culture having a chambervolume that is configured to house an immune cell culture, a satellitevolume for increasing the working volume of the chamber by providingadditional volume for media and other working fluids without housing theimmune cell culture, wherein the satellite volume is fluidly connectedto the cell culture chamber via one or more fluidics pathways such thatmedia is exchanged with the culture chamber without disturbing theimmune cell culture.

Embodiment 167 includes the cassette of embodiment 166, wherein thesatellite volume is a bag.

Embodiment 168 includes the cassette of embodiment 166, wherein thesatellite volume is a non-yielding chamber.

Embodiment 169 includes the cassette of embodiments 166-168, wherein thesatellite volume is further configured to allow media removal withoutloss of cells of the immune cell culture.

Embodiment 170 includes the cassette of embodiments 166-169, furthercomprising a crossflow reservoir.

Embodiment 171 includes the cassette of embodiments 166-170, wherein thecell culture chamber has a volume of between about 0.50 ml and about 300ml.

Embodiment 172 includes the cassette of embodiment 171, wherein the cellculture chamber has a volume of between about 50 ml and about 200 ml.

Embodiment 173 includes the cassette of embodiment 172, wherein the cellculture chamber has a volume of about 180 ml.

Embodiment 174 includes the cassette of embodiments 166-173, wherein thesatellite volume is between about 0.50 ml and about 300 ml.

Embodiment 175 includes the cassette of embodiment 174, wherein thesatellite volume is between about 150 ml and about 200 ml.

Embodiment 176 includes the cassette of embodiments 166-175, wherein thecrossflow reservoir has a volume of between about 0.50 ml and about 300ml.

Embodiment 177 includes the cassette of embodiments 176, wherein thecrossflow reservoir has a volume of between about 100 ml and about 150ml.

Embodiment 178 includes the cassette of embodiments 166-177, wherein theworking volume is about 180 mL to about 1 L.

Embodiment 179 includes the cassette of embodiment 178, wherein theworking volume is about 180 mL to about 460 mL.

Embodiment 180 includes the cassette of embodiments 157-179, wherein oneor more of the fluidic pathways comprise a silicon-based tubingcomponent that allows oxygenation through the tubing component.

EXAMPLES Example 1—Automated Production of CAR T Cells Using the COCOONSystem

In this Example, GFP and HER-2 lentivirus were used to transduce T cellsusing the following process parameters: starting inoculation of 60million peripheral blood mononuclear cells (PBMC), CD3/CD28 activation,IL-2 and IL-7 were supplemented into T-cell growth media for cultureexpansion. Single-use sensors in the disposable cassette were used tomonitor temperature, pH and optical density (OD) in real time. Themultiple cassette chambers that are connected via fluidic channelsenabled automated feeding and addition of process components. Some ofthe chambers are temperature controlled at 4° C. for media and reagentstorage, while others included elements for warming, mixing, washing,and concentrating cells, allowing for a fully enclosed process. Thein-process samples were drawn for cell counts and viability. At the endof the harvesting process, FACS analysis was performed with thefollowing panel: CD4, CD8, NGFR, IFN-γ, TNF-α, etc. An overview of theCOCOON System used in this Example is shown in FIG. 6. FIG. 6A shows theCOCOON system in the closed configuration along with an external usercontrol display, which can be used to adjust parameters or monitor thecell culture. Sterile, single-use cell culture “cassettes” can be loadedinto the COCOON (FIG. 6C). As shown in a detailed view of the cassette(FIG. 6B), each cassette includes an upper chamber maintained at 37° C.for growing cells, and a lower chamber maintained at 4° C. for storingmedia, viral vector, and other temperature-sensitive reagents. Thecassette is configured such that fluids can be exchanged through theinterior fluidics pathways, and also pumped into or out of the cassette.Sensors installed in the cassette can monitor, e.g., the pH and opticaldensity of the cell culture.

Results are shown in FIGS. 7-10. FIGS. 7A, 7B, and 7C show,respectively, the average harvest yields, average harvest viability, andaverage transduction efficiency for GFP transduction using the automatedCOCOON System, compared with manual manipulation and expansion of thecells using the G-REX (WilsonWolf) cell culture plates as control. TheG-REX plates have gas-permeable bottoms, and media exchange is typicallyperformed by the user every 4 to 5 days when using the G-REX.

FIGS. 8A and 8B show, respectively, the viable cells and the viabilityand transduction efficiency for HER-2 CAR-T transduction. In 10-daycultures, the HER-2 CAR-T cells reached approximately 2.2 billion withviability of 97% and transduction of 65% (n=4) in the COCOON system.

Performance of the automated COCOON System was also compared with manualmanipulation and growth of cells using the PERMALIFE Cell Culture Bag(OriGen) as a control. The PERMALIFE Bag is a sealable and gas-permeablecell culture bag made of inert fluorinated ethylene propylene (FEP),with valves to facilitate cell feeding and harvest by the user. FIG. 9Aindicates the relative T Cell purity level using the COCOON Systemcompared with the PERMALIFE Bag, as assessed by the percentage of CD3+cells. FIG. 9B shows a greater percentage of CD8+ cells cultured in theCOCOON System compared with the PERMALIFE Bag control. FIGS. 9C and 9Dshow that transfected cells produce TNF-α and INF-γ, respectively.

FIGS. 10A and 10B show effective and specific killing of target tumorcells by CAR T cells cultured in the COCOON System and the PERMALIFEBag, respectively.

In conclusion, the COCOON System, a fully enclosed cell engineeringsystem, is a viable solution to translate the labor-intensive CAR Tprocess into a fully automated and highly controlled system, thusallowing scalability, high yield, reduction of manufacturing cost, andgaining better process control to yield high quality CAR-T cells.

Example 2—Comparison of Activation Methods in the COCOON System

This Example compares cell culture performance using different methodsof activation in the clinical scale production of CAR T cells in theCOCOON automated manufacturing system and a PERMALIFE Bag.

Magnetic anti-CD3/anti-CD28 DYNABEAD activator beads may be used toactivate T cells. These beads provide the two necessary stimulatorysignals to support effective T cell activation. Another method ofactivating naïve T cells may utilize a soluble anti-CD3 antibody (OKT3).OKT3 is a monoclonal IgG2a antibody, originally used as animmunosuppressant. The costimulatory signals can be provided byaccessory cells. Initiating T cell culture from a mixed population ofperipheral blood mononuclear cells (PBMC) can provide the necessaryaccessory cells to support T cell activation when using OKT3.

As OKT3 and DYNABEADS utilize distinct activation mechanisms, theselection of one method over the other could influence the final productcharacteristics; specifically, the ratio of T cell subsets, CD4+ helperT cells and CD8+ cytotoxic T cells. The cytotoxic CD8 T cells areresponsible for the anti-tumor response. CD4 cells produce cytokines andhelp to regulate the immune response. It has been demonstrated that CD4cells also support cell lysis, although the killing is delayed comparedto CD8 cells. CD4 cells signal to APCs, thus activating APCs andsubsequently priming naïve CD8 T cells. The ideal target ratio of CD8 toCD4 cells is not well understood due to limited clinical data. Studieshave shown that a combination of CD8 and CD4 cells are preferred overthe delivery of CD8 cells alone (see, e.g., Church 2014; Feldmann 2012;Reusch 2015).

There are advantages and disadvantages of both methods of in vitroactivation. Antibody-bound beads offer consistency and ensure stablesimultaneous activation of the TCR/CD3 complex as well as the CD28co-stimulatory pathway. A major disadvantage of the bead approach is thehigh cost associated with this product. The beads must also beeffectively removed from culture before implantation. OKT3 offers alow-cost option for activating T cells. The major disadvantagesassociated with the soluble anti-CD3 approach are the dependency onaccessory cells and sensitivity to the culturing environment. Patientsamples may have highly variable accessory cells and negativeinteractions that might functionally inactivate the T cells afterprevious stimulation. To understand the impact of each method ofactivation on the growth, phenotype and functionality of the cells, Tcells activated by DYNABEADS and OKT3 were cultured in a clinical-scaleautomation platform.

COCOON provides the environmental control of gases and temperatures.This includes a 37° C. zone as well as a linked refrigerated zone. Thereis no fluid contact between the COCOON and the Cassette, minimizing therequired cleaning between runs. All reagents can be loaded into theCassette on the day of seeding and stored in the refrigerated zone ofthe COCOON until needed. Fluid is warmed to 37° C. before delivery tothe cells. Due to the stability of lentivirus, this can be thawed on theday of transduction and delivered into the Cassette via sterileconnectors. Gas exchange (oxygenation and CO₂ buffering) is achieved viarecirculation of the culture fluid through gas permeable tubing.Embedded biosensors provided real-time data on dissolved oxygen and pH.As the T cells require stable contact with other cells or the activatingagent, media exchanges, washing and recirculation for gas exchange canbe performed via perfusion without disturbing the cells. Rocking can beused to facilitate efficient harvesting.

Methods

Cell Culture. Peripheral blood mononuclear cells (PBMC) (Lonza) werethawed with DNase (Sigma) and allowed to recover overnight at 37° C. ata density of <2×10⁶ cells/mL. Cell counting was performed using theNUCLEOCOUNTER 200 with the Blood Assay protocol, including Solution 17(Chemometec). A third-generation lentiviral vector, encoded with a lowaffinity nerve growth factor receptor (NGFR) as a marker oftransduction, was used to transduce the cells. This lentivirus wasmanufactured at Lonza's cGMP virus manufacturing facility (Houston,Tex.) based on a protocol and primers originating from the Bramson Labat McMaster University (Hamilton, Canada). A multiplicity of infection(MOI) of 1 was used in all conditions. The viral titer was determined byusing HEK293™ cells and detection of NGFR using flow cytometry.Activation media consisted of X-VIVO 15 (Lonza) supplemented with 22IU/mL IL-2 (Cedarlane) and 1% penicillin-streptomycin (Sigma). Inconditions activated with soluble anti-CD3, OKT3 (Biolegend) was addedto the activation media for a final concentration of 50 ng/mL. Inconditions activated with DYNABEADS, a ratio of 1:1 beads to cells wasadded to the activation media. Expansion media consisted of X-VIVO 15(Lonza) supplemented with 29 IU/mL IL-2 (Cedarlane), 5% human serum frommale AB plasma (Sigma), 1% GLUTAMAX (Thermo Fisher) and 1%penicillin-streptomycin (Sigma).

Automated CAR T Cell Production. On Day 0, 60×10⁶ PBMC were loaded intothe input bag of the Cassette. In conditions activated usinganti-CD3/anti-CD28 beads, 60×10⁶ anti-CD3/anti-CD28 DYNABEADS(ThermoFisher) were also added to the input bags for a ratio of 1:1beads to cells. The input bag was connected to the Cassette and broughtto the COCOON (Octane Biotech Inc.). Following operator sign-in, theCassette was loaded into the COCOON. On Day 1, virus (Lonza Houston) wasthawed and then transferred to the cell culture chamber via the Cassetteaccess port at a MOI of 1. Prior to delivery of the virus to the cells,activation media was used to dilute the media. The activation media wasremoved from the culture chamber and returned with the virus withoutdisturbing the cells. The total working volume was increased on Day 4with the addition of expansion media. Partial media exchanges wereperformed with expansion media on Day 6 and Day 8. Following theexpansion steps, the COCOON decreased the final volume to less than 100mL before the cells were removed. Throughout the culture, data wascontinuously collected by the COCOON. This included every pump andactuator step, each time the door was opened and closed and so forth.Comprehensive sensor data was collected including thermal values, gasconcentrations, fluid pH and dissolved oxygen. The operator was able toremotely monitor the status of the culture using a phone or externalcomputer.

Manual CAR T Cell Production. Manual production of CAR T cells wasperformed in parallel to COCOON in PERMALIFE cell culture bags. On Day0, 60×10⁶ PBMC were seeded in activation media at 0.27×10⁶ cells/mL onDay 0. These cultures utilized the same donor cells as well as the samemedia for activation and expansion as the automated cultures. Cultureswere initiated in PERMALIFE bags (PL240, Origen) and were transferred tolarger PERMALIFE bags on Day 6 (PL325, Origen) as the cells expanded.Cells were expanded into PL240 and PL325 bags on Day 8 as the volumeincreased. On Day 1, lentivirus was added to the bags at a MOI of 1.Cells were fed with an equivalent volume as COCOON cultures; however,unlike the COCOON conditions, no media was sent to waste. The volumeused maintained the cultures at less than 2×10⁶ cells/mL. On Day 10,culture volumes were obtained by mass and a sample of the total cellswas removed from the bags for counting and analysis. The cells werecentrifuged to reduce the residuals as well as the volume before use infunctional assays.

Non-Transduced and Non-Activated Conditions. Non-transduced andnon-activated negative controls used for fluorescence activated cellsorting (FACS) analysis were cultured at a small scale according toprotocols previously described. Briefly, 1×10⁵ cells were seeded in 96well plates with X-VIVO 15 (Lonza) supplemented with 5% human AB serum(Sigma) and 22 ng/mL IL-2 (Cedarlane). Activated, but non-transducedcontrols were set up using a similar protocol. After the cells wereseeded, an equal volume of media was added. Conditions activated withsoluble anti-CD3 were supplemented with 100 ng/mL OKT3 (Biolegend) for afinal concentration of 50 ng/mL. Conditions activated withanti-CD3/anti-CD28 beads had DYNABEADS added at a ratio of 1:1.Activated cultures were expanded from 96 well plates to 24 well on Day 4and transferred into T25 and T75 flasks based on their growth and fedevery two days from Day 4.

Flow Cytometry. To phenotype starting populations, cells were stainedwith the following primary antibodies: Pacific blue CD3 (clone UCHT1, BDBiosciences), PE CD14 (clone 61D3, ThermoFisher), APCeFluor780 CD4(clone OKT4, ThermoFisher), PerCP-Cy5.5 CD8a (clone RPA-T8,ThermoFisher), BV605 CD279 (PD-1, clone EH12.2H7 BioLegend) andLIVE/DEAD Fixable Violet Dead Cell Stain (ThermoFisher). To assess theefficiency of HER2 transduction, cells were stained as above exceptinstead of staining for monocytes (CD14), cells were stained with BV421CD271 (C40-1457 NGFR, BD Biosciences) and LIVE/DEAD Fixable Green DeadCell Stain (ThermoFisher). Cells were then fixed and washed. Greaterthan 20,000 events were acquired per condition on a SA3800 Sony SpectralAnalyzer. FACS analysis was performed using FlowJo 10.4.2.Non-transduced and non-activated conditions were used to set gates alongwith fluorescence minus one (FMO) controls.

Tumor Cell Lines. HER2 negative tumor cells, LOX-IMVI cells (NationalCancer Institute), derived from metastatic amelanotic melanoma wereexpanded in RPMI (Sigma) with 10% FBS (Sigma) as previously described.HER2 positive tumor cells, SKOV-3 (ATCC) cells, derived from an ovarianserous cystadenocarcinoma were expanded in McCoy's 5a (modified) media(ThermoFisher) with 10% FBS as previously described. Cells were passagedbefore confluence using 0.25% trypsin for 5 to 10 minutes. Low passagenumbers were cryopreserved and tumor lines were passaged 2 to 3 timesbefore use in ALAMARBLUE or ICS assays.

Cytokine Secretion Assay. As previously described (e.g., Atkuri 2005;Avgoustiniatos 2008), 50,000 LOX IMVI or SKOV-3 tumor cells were seededin triplicate for each culture condition into round bottom 96 wellplates. The following day, T cells were seeded at 8:1 per well of thetumor lines with a protein transport inhibitor brefeldin A (Golgi Plug,BD Biosciences) for 4 hours at 37° C. Cells were stored at 4° C.overnight. Cells were then pooled for staining and analysis. Asdescribed above, cells were stained for surface phenotype CD3, CD4,CD8a, NGFR, and LIVE/DEAD Fixable Green Dead Cell Stain. Intracellularcytokine staining (ICS) was completed following fixation andpermeabilization with BD Cytofix/Cytoperm Fixation/PermeabilizationSolution Kit (554714, BD Biosciences). Activated cytokines testedinclude APC IFNγ (clone B27, BD Biosciences) and PE TNFα (clone MAb11,BD Biosciences). More than 230,000 events (maximum 500,000) werecollected on the Sony SA3800 for ICS analysis. The difference betweenproduction of cytokines on SKOV-3 and LOX-IMVI tumor lines was reportedas the percentage of the population secreting TNFα or IFNγ.Non-transduced and non-activated conditions were used to set gates alongwith FMO controls.

Cytotoxicity Assay. Cytotoxicity was tested as previously described(e.g., Atkuri 2005; Avgoustiniatos 2008). Adherent tumor cell lines wereplated at 2×10⁴ cells/well (SKOV-3 or LOX-IMVI) overnight in 96-wellflat bottom tissue culture treated plates. CAR T cells from the COCOONand control conditions were added to wells of tumor cells at variouseffector (E) T cells to tumor (T) E:T ratios (from 0.25:1 to 8:1) andco-incubated overnight at 37° C. Wells were washed three times withwarmed PBS or RPMI media to remove any non-adherent cells. 100 μL of a10% solution of ALAMARBLUE cell viability reagent (Life Technologies)was added and wells were incubated at 37° C. for 3 hours. ALAMARBLUE, ametabolic indicator of viable cells that fluoresces upon mitochondrialreduction, was measured by fluorescence (excitation 530 nm, emission 595nm) on a Tecan Infinite M200 Pro plate reader (Tecan, Maennendorf,Switzerland). Tumor cell viability was calculated as the loss offluorescence in experimental wells compared to untreated target cells.Each condition was tested in triplicate.

Results

The automation platform, COCOON, was utilized to demonstrate thefeasibility in achieving clinical-scale production of CAR T cells usingtwo different activation methods. The platform consists of a single-usedisposable COCOON Cassette (FIG. 11A, 11E) and a COCOON control system(FIG. 11B). FIG. 11F shows how a syringe 1170 or bag 1172 can be usedfor cassette 602 sampling. The cassette is designed with multiplereagent bags to enable all reagents required for the process to bepre-loaded and stored in the refrigerated zone of the cassette with cellprocessing occurring in the culture zone. The cassette supports multipleunit operations linked as a closed system, including cell activation,transduction, expansion, real-time dissolved oxygen and pH monitoring,washing, and cell concentration. The lower portion of the Cassettecontains multiple bags to hold the various reagents and waste requiredfor the culture. COCOON provides the control system for cassettes. Thisincludes control of fluid and cell transfers, as well as rocking,agitation and remote monitoring of control sensors. Actuators enableautomated valve control without fluid contact. Without actuatorinteraction, valves remain closed enabling the cassette to be movedbetween rooms or to a microscope while preventing uncontrolled fluidmovement. After loading the required reagents into the fluid reservoirof the Cassette, it is snapped on to the culture zone of the Cassette inwhich various unit operations occur. Sterile sample removal or injectionof virus utilizes ICU Spiros connectors. Prior to sample removal orvirus addition, the operator is promoted at a specific time, as definedin the pre-programmed protocol. Following operator sign-in andacknowledgment of the notification, the COCOON automatically opens toenable sample removal or virus addition. The operator acknowledges thatthe action has been completed before the door automatically closes andenvironmental control resumes. When the Cassette is loaded into theCOCOON (FIG. 11C) and the outer shell is closed (FIG. 11D), the lowerportion of the Cassette is separated from the upper portion by a thermalbarrier. The lower portion is maintained at refrigerated temperaturesand the upper portion is maintained at 37° C. The closed COCOON enablesgas and thermal control. Cells are maintained at 37° C. while reagentsare maintained in a cold zone to prolong stability. The opaque shellprevents light-induced toxicity related to the breakdown of mediacomponents. A pre-warming chamber is located in the 37° C. zone to warmmedia before it is transferred to the cells. All culture steps can beautomated from the PBMC loading to the final concentration and cellcollection. As shown in FIG. 11A, the Cassette has a series of accessports which can be used for loading the virus following activation. Realtime dissolved oxygen and pH sensors are incorporated into the Cassetteto provide feedback to the COCOON software. Real time data as well ashistorical graphs can be monitored to ensure that these factors weremaintained within the target ranges.

An overview of the COCOON process steps is shown in FIG. 12A. Gaspermeable PERMALIFE bags were used for parallel control cultures and theexpansion of CAR T cells (e.g., Lu 2016). FIGS. 12B (COCOON) and 12C(PERMALIFE bag) demonstrate the cell distribution in the two formatswith the cells in the COCOON cultured in the top chamber of Cassette. Anequivalent volume of media was used for both systems. The PERMALIFE cellculture bag utilized a fed batch process, with the area expanded astotal volume increased, as is commonly performed. The COCOON Cassetteutilized a fixed area, employed an initial fed batch feeding strategyand then used partial media exchanges on Day 6 and Day 8 of culture.

To assess the impact of the activation method and the performance of theautomated platform, the following criteria were used: viability, cellnumber, phenotype, exhaustion, transduction efficiency, functionalintracellular cytokine secretion, and cytotoxicity. Results aresummarized in FIG. 16 and discussed herein.

The same donor cells were used for all conditions, unless otherwiseindicated as Donor 2. All conditions were seeded with 60×10⁶ PBMC andfed with the same media volume and composition. The starting cellpopulation contained 66.6% CD3+ T cells and 12.0% CD14+ cells. Of theCD3+ cells, 71.2% were CD4+ and 28.1% were CD8+ cells. A second donorwas used to determine the impact of donor-to-donor variability. Thissecond population of PBMC originally contained 75.0% CD3+ T cells and4.5% CD14+ cells. Of the CD3+ cells, 65.0% were CD4+ and 32.9% were CD8+cells.

The Day 10 viable cell yield from the COCOON cultures activated withOKT3 and DYNABEADS were 2.55×10⁹±0.1×10⁹ and 2.15×10⁹±0.1×10⁹respectively. The viable cell yield from the PERMALIFE bag culturesactivated with OKT3 and DYNABEADS were 2.08×10⁹±0.1×10⁹ and1.53×10⁹±0.1×10⁹ respectively (FIG. 13A). The viability in allconditions was greater than 95% (FIG. 13A). The population doublinglevel (PDL) was 5.2-5.4 in COCOON (36-43 fold) and 4.7-5.1 in thePERMALIFE bags (25-35 fold) (FIG. 13B).

All conditions exhibited a high level of purity of T cells, with greaterthan 88% of the viable cells expressing CD3. The total viable T cellsgenerated in 10 days was greater than 2 billion, with the exception ofbead activated PBMCs grown in the PERMALIFE bags (FIG. 13C). Regardlessof the activation method, the total T cell yield was greater in theCOCOON conditions compared to the bags. The Day 10 COCOON Cassette Tcell yield was 2.0-2.4×10⁹. The PERMALIFE bags produced 1.5-2.0×10⁹ Tcells (FIG. 13C). Using the same donor cells, the PDL of CD3+ cells was5.7 and 5.9 (51 and 60 fold) in COCOON activated using DYNABEADS or OKT3respectively (FIG. 13D). The PDL of the CD3+ cells was 5.2 and 5.6 (38and 49 fold) in the PERMALIFE bags activated using DYNABEADS and OKT3respectively.

The percentage of CD3+ T cells expressing CD4 and CD8 glycoproteins,indicative of helper or cytotoxic T cells respectively, are shown inFIG. 13E. The most significant result related to the T cellsubpopulations was the increased number of CD8 cells in the conditionsactivated with OKT3 compared to the DYNABEAD-activated cells. OKT3activation resulted in 83-86% CD8+ and 6-11% CD4+ cells while DYNABEADactivated conditions resulted in subpopulations of 48-56% CD8+ and41-48% CD4+ cells. In all cultures with the same donor, the exhaustionassociated marker, PD-1 was below 10%, indicating low levels of cellexhaustion (FIG. 13F). The second donor expressed PD-1 in 21% of thecells when cultured in COCOON with DYNABEADS. FIGS. 13G and 13H showrepresentative contour plots highlighting the significant difference inCD8+ cells in the DYNABEAD-activated conditions compared to theOKT3-activated conditions.

High transduction efficiency was determined by surrogate surface markerCD271 (NGFR) expression for T cell HER2 specificity with 62-78% of CD3+cells in COCOON and 42-60% of CD3+ cells in PERMALIFE bags expressingNGFR (FIG. 14A). The transduction efficiency was greater in the COCOONcompared to the bag cultures. With the high transduction and expansion,the total number of viable CAR T cells ranged from 1.26-1.66×10⁹ inCOCOON and 0.62-1.20×10⁹ in PERMALIFE bags (FIG. 14B). The percentageand total number of CAR T cells in the CD4 and CD8 subpopulations areshown in FIGS. 14C and 14D respectively. The percentage of transducedCD4 cells was greater than the CD8 cells with 75.4-80.9% of CD4 cellsand 64-73.2% of CD8 cells in COCOON expressing NGFR. In the PERMALIFEbags, 54.7-79.9% of the CD4 cells and 36.1-58.9% of the CD8 cellsexpressed NGFR. As the expansion of the CD8 cells was significantlygreater than the CD4 cells, the total number of CD8+ transduced cellswas significantly greater than the CD4+ transduced cells in allconditions except DYNABEAD-activated bag cultures (FIG. 14D). In theCOCOON there were 0.25-0.64×10⁹ transduced CD4 cells and 0.66-1.43×10⁹transduced CD8 cells. In the PERMALIFE bag conditions, there were0.09-0.41×10⁹ transduced CD4 cells and 0.25-1.06×10⁹ transduced CD8cells. Representative contour plots of the transduction efficiency inthe COCOON conditions and PERMALIFE bag conditions are shown in FIGS.14E and 14F respectively.

Functionality testing of the cells was performed using an intracellularcytokine release assay and an ALAMARBLUE killing assay (see Nociari1998) (FIG. 15). In all cases, the cells demonstrated production of TNFαand IFNγ (FIGS. 15A and 15B), characteristic of type 1 T helper CD4+cells and cytotoxic CD8+ cells (see, e.g., Romagnani 1991). Higherproportions of CD4+ cells secreted TNFα. The production of TNFαsecreting cells was greater in the COCOON conditions compared to the bagcultures for the same donor cells. The DYNABEAD-activated conditionsproduced higher percentages of TNFα and IFNγ secreting transduced cellsthan the OKT3-activated conditions. The ALAMARBLUE killing assaydemonstrated effective killing of ovarian carcinoma cell line SKOV-3HER2+ tumor cells by the CAR T cells (FIGS. 15C and 15D). The trends ofkilling effectiveness followed the serial dilution of the effector Tcells with strong response from both PERMALIFE and COCOON generatedcells. HER2− tumor cells, LOX IMVI, were also exposed to the T cells todemonstrate HER2 specificity. No killing trends were identified in theHER2 negative cultures in response to the CAR T cells.

Discussion

Activation Method. Assessment of CAR T cell production includedactivation using soluble anti-CD3 (OKT3) as well as the bead-boundanti-CD3/anti-CD28 DYNABEADS. The cultures activated with OKT3demonstrated improved growth of 19-36% over DYNABEAD-activated cultures(FIG. 13A). The method of activation also generated a significantdifference in the final phenotype (FIG. 13E). The DYNABEAD-activatedconditions had an average of 52.7% CD3+CD8+ cells compared toOKT3-activated conditions, which had 84.5% CD3+CD8+. This represents aCD8+ to CD4+ ratio of approximately 1.2:1 for DYNABEAD-activatedconditions compared to 9.8:1 when activated with OKT3. The increasednumber of CD8+ cells were found regardless of whether the cells werecultured in bags or COCOON conditions.

The improved yield with OKT3 activation was an unexpected result.DYNABEADS activate T cells by binding to the TCR/CD3 complex as well asthe CD28 co-stimulatory receptor. Unlike DYNABEADS, which have ananti-CD28 antibody for co-stimulation, activation with soluble anti-CD3relies on monocytes to present B7 receptors, CD80 and CD86, which areligands to CD28 (see, e.g., Fleischer 1996). However, the B7 receptorscan also bind to CTLA-4 and stimulate this inhibitory pathway, thusinhibiting T cell growth. The improved total cell yield based onactivation method was found regardless of whether the cells werecultured in bags or COCOON conditions. Bead-bound anti-CD3/anti-CD28antibodies may promote the expansion of helper T cells (CD4+ cells)while OKT3 may promote the expansion of cytotoxic T cells (CD8+ cells)(see, e.g., Fleischer 1996; Laux 2000; Li 2010; Zhu 2007).

The higher cell yield, and specifically, the CD8+ cell predominance maybe attributed to the stimulation of additional receptors when activatedusing OKT3 and monocytes. It has previously been reported that 95% ofCD4+ T cells express CD28 while only 50% of CD8 cells express CD28 (seeLedbetter 1990). Consequently, DYNABEADS may only activate a maximum of50% of the CD8+ cells. The cultures activated with OKT3 may benefit fromother co-stimulatory ligands that are present on the monocytes and noton the beads.

For example, monocytes express CD58 (LFA-3) and CD40 receptors, whichare ligands for CD2 and CD40L. Stimulation of these receptors is knownto promote T cell growth. These accessory cells may also express CD137L,which interacts with CD137 and may stimulate CD8+ cell expansion. Theinteraction with these other receptors may representative a morephysiologic antigen presentation compared to DYNABEAD activation.

As OKT3 activation is dependent on other cells, the impact of donorvariability may be more significant than activation with DYNABEADS. Thestarting cell population in this study was comprised of 12.0% CD14+cells and 66.6% CD3+ cells on Day 0. A dose study could be performed todetermine the impact of monocyte-sensitivity on the final yield andphenotype.

Automation. The COCOON generated a greater yield of viable CAR T cellscompared to the manual conditions when activated with either OKT3 orDYNABEADS. When activated with DYNABEADS, the COCOON cultures yielded40% more growth than bag cultures. With OKT3 cultures, the COCOONyielded 23% more cells than bag cultures. The COCOON conditions alsodemonstrated greater transduction efficiency and consequently a greatertotal yield of CAR T cells (FIG. 14). With DYNABEAD-activatedconditions, the total CAR T cell yield in the COCOON was more thandouble that of the bags. With OKT3-activated conditions, the yield ofCAR T cells was approximately 40% more in COCOON than the bags.

The improved yield in COCOON over the PERMALIFE bags may be increasedactivation. This may be due to the distribution across culture area. TheCOCOON utilizes a solid non-yielding chamber whereas the bags areflexible. Following cell settling, it was observed that the curvature ofthe bag caused an uneven distribution of cells. This may have caused anuneven distribution of activation agent and/or cells. Another possiblecause may be related to the amount of agitation during the activationphase. As the cells were transduced the day after activation, activationmay still have been in progress or the activation agent may not havebeen internalized by the cells. During the transduction step, the bagcultures are moved from the incubator to the biosafety cabinet todeliver the cells using sterile technique. The movement of the bagsfacilitates virus distribution in the bags; however, the cells are alsodisturbed during the transfer of the bags to and from the incubator. Asstable contact may be important for cell activation, this movement mayhave negatively impacted the cells. The cells in the COCOON cultures arenot disturbed between the activation or transduction step. In theCOCOON, the media used for activation is removed from the culture priorto transduction. A small volume of media is left in the chamber thatenables the cells to remain at the bottom of the chamber, undisturbedduring the volume transfers. The media removed from the chamber is usedto dilute and mix the virus and is then transferred back to the cellpopulation. During this process, the cells remain undisturbed.

Efficient activation may correlate to more efficient transduction. Thatis, if the cells are activated and are actively dividing, the lentiviruscould integrate more effectively. To assess this, samples could be takenprior to transduction to determine the activation efficiency. Theimproved transduction efficiency may also be related to the homogeneousdistribution of the virus to the cells. In COCOON cultures, the virus ismixed with the media and uniformly distributed to the cells. Using aflat, non-flexible, vessel helps to improve homogeneous distribution andconsequently homogeneously exposure of the virus amongst the cellpopulation.

Another reason for the improved performance may be related to gasexchange. Increased oxygen levels may support increased proliferation.High oxygen levels were maintained in the automated platform by usingrecirculation of the culture supernatant through a silicone gas exchangeline. Gas exchange is achieved in the bag conditions by diffusionthrough the bag material, fluorinated ethylene propylene (FEP). Thepermeability coefficient of silicone is significantly greater than thepermeability of the FEP (see, e.g., Avgoustiniatos 2008). The COCOONprotocol was created to ensure sufficient oxygen concentration. This wasconfirmed by biosensor data generated throughout the culture period.

The gas exchange via the silicone tubing also supports pH level. Thatis, at the beginning of the culture, the media maintains the target pHby gas exchange with a CO₂ enriched environment. As the cell number inthe culture increases, the cells produce lactic acid and CO₂, to removethe need for a CO₂ environment. The CO₂ in the COCOON environmentdecreased over the culture duration to help to maintain pH. ThePERMALIFE bags followed a conventional protocol of being stored in a 5%CO₂ environment throughout the culture process.

An additional advantage of the continuous recirculation, withoutdisturbing the cells, is a more homogeneous distribution of positive andnegative factors. This includes nutrients, waste, released cytokines anddissolved gases. Continuous recirculation may help to reduce localizedeffects and improve the media efficiency by evenly distributing factors.

Automation Translation. In this Example, a closed and automatedproduction system, COCOON, was used to generate CAR T cells activated byeither bead-bound antibodies or soluble OKT3. The results demonstratethat a clinically-relevant yield can be generated from COCOON with ahigh transduction efficiency using a low concentration of virus.Furthermore, the phenotype of the cells can be driven by the activationmethod.

The results were primarily generated from a single donor to compare theimpact of activation method. The variability between conditions was verylow. When the test was repeated with a different donor, the results weresimilar between donors when using the same method of activation. Thisstudy demonstrates an efficient method of effectively automating theproduction of CAR T cells in a clinically-relevant, scalable, and easyto use method.

Example 3—Transduction Via Electroporation with a Cell EngineeringSystem Background

The Octane Cocoon™ system is an automated, closed, end-to-end bioreactorsystem for the manufacture of cell therapy products. Octane's AutomatedCell & Tissue Engineering System (ACTES) is comprised of three maincomponents: the base instrument, software, and customizable disposablecassette. The Cocoon™ system is capable of automated isolation,expansion, concentration, and buffer exchange for both upstream anddownstream cell culture processes.

An electroporation unit enables transfection of cells traditionallyknown to have low transfection efficiency via electroporation and othernon-viral methods, including primary cells, stem cells, neurons, andresting or non-proliferating cells. The system includes anelectroporation unit, electroporation solutions, electroporationCartridges and optimized electroporation protocols. The electroporationunit is comprised of a Core Unit and 1-3 additional functional add-onunits addressing different needs. For example, the electroporation unitcan be used to transfect varying cell numbers in 20 μL-100 μL and 1×10⁷to 1×10⁹ in 1 mL-20 mL volume.

Described herein is an automated, completely closed, sterile and robustTransfection and cell expansion procedure using an electroporation Unitand Octane Cocoon™ systems. In the proof-of-concept (PoC) evaluations,the respective electroporation Software and Octane Cocoon™ ACTESsoftware will operate independently of one another. In otherembodiments, the software is fully integrated between the systems.

Methods

Evaluation of Peripheral Blood Monocyte Cell (PBMC) transfection andexpansion using the electroporation unit and Cocoon™ systems was dividedinto three main focus areas:

Cell concentration in the Cocoon™ cassette, cell transfer between theOctane Cocoon™ and the electroporation Unit, expansion of transfectedcells transferred between the Cocoon™ and electroporation Unit and cellconcentration in the Cocoon™ cassette.

The Cocoon™ ACTES cassette recirculates about 450 mL of culture media inits culture chamber. The cell proliferation chamber typically holds aconstant volume of up to 180 mL of media within its 260 cm² area.Additional media volume beyond the 180 mL capacity of the 260 cm²proliferation chamber is provided from various satellite reservoirs andchambers of the Cocoon™ cassette. The additional media from thesesatellite reservoirs can be recirculated within the culture portion ofthe disposable Cocoon™ to provide fresh nutrients and remove wasteproducts from cells in the 260 cm² proliferation chamber.

An exemplary volume that the electroporation Unit can transfect is 20mL. The 20 mL volume should suitably be comprised of at least 90% of theappropriate electroporation Solution. Thus, for PoC studies, theoriginal culture volume was reduced to 10 mL, then diluted in anadditional 90 mL of supplemented P3 Primary Cell electroporationSolution, and concentrated to a final volume of 10 mL-18 mL.

The Proof of Concept studies described utilized the following:

A 20 gauge, 0.024″ I.D./0.036″ O.D., flow restrictor from Nordson EFD,which was added to the end of the permeate line.

1×10⁸ PBMCs were stimulated with 1×10⁸ CD3+:CD28+ Dynabeads (Invitrogen)and expanded in Complete T-cell Media comprised of X-VIVO 15 media(Lonza) supplemented with 5% Human Serum A/B (Sigma) and 10 ng/mL IL-2(Peprotech) using multiple GREX 100 (Wilson Wolf) culture vessels for upto 10 days. Test concentrations of cells were transferred to 250 mLconical vials and allowed to settle in 37° C. incubators with 5% CO2 inair humidified for 2-4 hours. The supernatant of the settled cellsuspension was reduced to 10 mL and excess supernatant discarded. 90 mLof supplemented P3 Primary Cell electroporation Solution (Lonza) wasadded to the concentrated cell suspension for a final volume of 100 mL.The 100 mL cell suspension was then concentrated to a volume of 10 mL. Acontrol sample of cells were incubated at 37° C.

Counts were performed in duplicate using the Nucleocounter NC-200(Chemometec) on the pre-diluted cell culture, the diluted culture andthe final concentrated cell suspension. Volumes were measured using aserological pipette and KrosFlo scales. Residual testing samples wereobtained from the initial culture pre-dilution, supernatant, and finalconcentrated cell suspension. A Human Serum ELISA Kit (BethylLaboratories) was used to determine the percentage of serum remainingpost dilution and concentration. FACS analysis was performed on controlcells and concentrated cell suspensions for CD4+ and CD8+ expression.

Successful demonstration of volume reduction for Cocoon™ transfectionprotocols was defined as follows: ≥85% recovery of cells, ≤0% decreasein cell viability and 0% residual human serum of the initialconcentration.

Cell Transfer between the Octane Cocoon™ and the electroporation Unit

The transfer of cells between the Cocoon™ and electroporation Unitrequires several disposable consumables: the Cocoon™ cassette, theelectroporation Cartridge, two modified electroporation Reservoirs, andtwo Connection Tubing Sets (See FIG. 17).

The modified electroporation Reservoirs include inlet and outletweldable tubing with a luer lock connection endings, a cell inlet portwithin the Reservoir housing connected to the external inlet Reservoirtubing for sterile cell transfer into the Reservoir, a luer locksubstrate addition port on the inlet tubing of the LV Reservoir, and avent filter on the cap for air escape during volume transfer. TheCocoon™ cassette is designed with a port capable of automating transferof fluids and cell suspensions outside of the Cocoon™ in a controlledmanner, without compromising the sterility or cellular health of theculture.

Successful demonstration of aseptic transfer of cells between theCocoon™ and electroporation Unit demonstrated: supernatant oftransferred, transfected cells passed sterility testing, no mycoplasmadetected in pre- and post transfected culture samples≥90% recovery ofpre-transfected cell/volume in the Cocoon™ cassette post transfectionand delivery to the Cocoon™ cassette proliferation chamber, ≤5% changein viability of non-transfected cells between Cocoon™ andelectroporation Unit cell transfer movements and ≤20% change in CD3+,CD4+, and CD8+ cells when comparing cells transfected with and withoutautomated transfers between the Cocoon™ and electroporation unit.

The Cocoon™ ACTES Cassette has two sampling ports with BD Q-Syte femaleluer lock endings, as well as inlet and outlet ports with cannulas thatallow for automated transfer of cell suspensions out of the Cocoon™cassette and through Connection Tubing Sets aseptically connected tothese locations. During PoC studies, connections between the Cocoon™cassette, electroporation Reservoirs, electroporation Cartridge, andConnection Tubing Sets were aseptically connected to produce a sterileloop between the Cocoon™ and electroporation systems as follows.

Connection Tubing Sets with ICU Medical Spiros® male luer lock endingconnectors were connected to the two BD Q-Syte female luer lock samplingports of the Cocoon™. To make a sterile pathway from the Cocoon™cassette to the electroporation Reservoir, the other Spiros® male luerlock connection (ICU Medical) of the Connection Tubing Set was connectedto the female luer lock inlet tubing of the electroporation Reservoir.To connect the modified electroporation Reservoir to the electroporationCartridge, the female luer lock ending of the modified electroporationReservoir drain line was attached to the Spiros® male luer lockconnection (ICU Medical) of the electroporation Cartridge inlet. Forcollection of the transfected cells, the Spiros® male luer lock outputconnection of the electroporation Cartridge was connected to the femaleluer lock connector inlet of a second electroporation Reservoir. Thefemale luer lock ending of the second electroporation Reservoir drainline was connected to the Spiros® male luer lock connector of theConnection Tubing Set on the second automated sampling port of theCocoon™ cassette.

In embodiments, the Cocoon™ pump transfers the transfected cells to theCocoon™ proliferation chamber, the second electroporation Reservoir orother collection vessel capable of aseptic transfer of cells is utilizedto collect the newly transfected cells before delivery to theproliferation chamber of the Cocoon™ cassette. Sterile weldingtechniques can be used in place of aseptic luer lock connections betweenthe inlet and outlet PVC tubing lines of the modified electroporationReservoirs and Connection Tubing Sets with PVC tubing is feasible.

The cell engineering systems (Cocoon) described herein also allows forsterile, closed connections between the Cocoon™ cassette and anelectroporation Unit, via tubing guided from the internal Cocoon™environment through a hollow shaft of the Cocoon™ instrument. Thishollow shaft, referred to as the “Trumpet Arm”, provides access to theinternal environment of the Cocoon™ culture chamber from the externalenvironment without loss of control over key process parameters. Cellmovement between the Cocoon™ and electroporation Unit used theperistaltic pumps and software of the two separate control systems, butcan also utilize software of a combined system to control the separatepumping systems.

Prior to transfection, cells/fluid were either manually transferred to asterile electroporation Reservoir to mimic pre-expansion (Day 0)transfection procedures or transferred from the Cocoon™ cassetteproliferation chamber to mimic post-expansion transfection procedures tothe sterile electroporation Reservoir using the Cocoon™ pump, software,and Connection Tubing Sets (previously described). The Cocoon™ pump andsoftware then automated the transfer of cells/fluid from the Cocoon™cassette to the inlet of the electroporation Reservoir. Theelectroporation system executed pre-programmed pump movements of up to20 mL from the electroporation Reservoir, through the electroporationCartridge, and to the second electroporation Reservoir. The Cocoon™ pumpthen transferred the collected transfected cells/buffer from the secondelectroporation Reservoir to the proliferation chamber of the Cocoon™cassette.

A second electroporation Reservoir was incorporated to collect thetransfected cells and hold them until ready to be transferred by theCocoon™ pump to the Cocoon™ proliferation chamber. To use only theelectroporation Unit pump to move the transfected cells from theelectroporation Unit to the Cocoon™ proliferation chamber, a “ConnectionTubing Set Clearing” program can be utilized. In addition, theConnection Tubing Sets should be consistent in length.

Using the Cocoon™ cassette, Connection Tubing Sets, and modifiedelectroporation Reservoir connections previously described (FIG. 17), 11mL of Phosphate Buffer Solution (Lonza) was transferred from the Cocoon™cassette to the modified electroporation Reservoir using the Cocoon™pump. An electroporation program was used to perform a mock transfectionof the PBS solution and move the 11 mL volume to the second modifiedelectroporation Reservoir. The Cocoon™ pump and software was then usedto transfer the 11 mL volume from the second modified electroporationReservoir to the output bag of the Cocoon™ cassette. Volume transferredto the satellite bag from the Cocoon™ reservoir was estimated at 11 mLper run. Actual volume was measured using serological pipette aftertransfer to the first modified electroporation Reservoir, secondmodified electroporation Reservoir, and Cocoon™ output bag. Passingcriteria was established at ≥90%, fluid recovery from the first modifiedelectroporation Reservoir to the Cocoon™ output bag.

Cell Suspension Testing

1×10⁸ and 5×10⁸ total viable PBMCs will be expanded in 450 mL ofComplete T-cell Media, comprised of X-VIVO 15 media (Lonza) supplementedwith 5% Human Serum A/B (Sigma) and 10 ng/mL IL-2 (Peprotech), in thesterile Cocoon™ ACTES cassettes. On day 3, 440 mL of the culturesupernatant will be removed and held for sterility and mycoplasmatesting. The cells will be diluted in 90 mL of supplemented P3electroporation Solution (Lonza). The cells will then be concentrated inthe Cocoon™ cassette to approximately 10 mL of cell suspension andtransferred to the Cocoon™ satellite bag. An option to wash theproliferation chamber with an additional 10 mL of supplemented P3electroporation Solution and added to the cell suspension in the Cocoon™satellite bag will be evaluated. A sample will be removed from theconcentrated cells in the satellite bag for duplicate cell counts usingthe Nucleocounter NC-200 (Chemometec), mycoplasma, and sterilityretains. The cells will then be transferred to a modifiedelectroporation Reservoir via the Cocoon™ pump and Connection TubingSets, as previously described. The electroporation Unit pump and EO-210program will be used to transfect the T-cells with pmax GFP Vector(Lonza) and transfer the transfected cells to a second modifiedelectroporation Reservoir. The Cocoon™ pump will then transfer the cellsfrom the modified electroporation Reservoir to the proliferation chamberof the Cocoon™ ACTES cassette. A sample of the cells will be removedfrom the ACTES cassette proliferation chamber for duplicate cell count,mycoplasma, and sterility testing. This procedure will be repeated witha control culture in which cells will not be transfected, but insteadpassed through the electroporation Unit using the mock electroporationProgram CA-100. This procedure will be evaluated using three differentdonors; both freshly isolated and from cryopreserved PBMC lots.

Change in cell viability will be measured in the non-transfected cellcultures. Cell recovery, sterility, and mycoplasma load will be assessedin all cultures. Flow cytometry will be used to evaluate GFP, CD3+,CD4+, CD8+, and additional marker expression.

Aseptic Transfer of Cell Suspensions between Cocoon™ and electroporationUnit provide sterile and mycoplasma-free supernatant pre- andpost-movements, ≥90% recovery of pre-transfected cells in the Cocoon™cassette post transfection and delivery to the Cocoon™ cassetteproliferation chamber, ≤5% change in viability of non-transfected cells,≤20% change in CD3+, CD4+, and CD8+ cell ratios post transfection.

Expansion of Transfected Cells Transferred Between the Cocoon™ andElectroporation Unit

1×10⁸ and 5×10⁸ total viable PBMCs will be expanded and concentrated inthe Cocoon™ cassette, transfected via sterile connection theelectroporation LV Unit, and sterilely transferred to the Cocoon™, asprevious described in the methods section for “Cell Transfer between theOctane Cocoon™ and the electroporation LV Unit, Cell SuspensionTesting”. Transfected cells will be cultured for up to 15 days in theCocoon™ cassette proliferation chamber using the most relevant andoptimized automated Cocoon™ protocol. A control will be expanded in aT-225 flask (Corning) or GREX 100 (Wilson Wolf) culture vessel for 3day. On Day 3, the control culture will be aseptically and manuallyconcentrated, transfected via the electroporation LV Unit EO-210program, and transferred back to the original vessel for continuedexpansion of up to 15 days. This procedure will be evaluated using threedifferent donors, from either freshly isolated or cryopreserved PBMClots.

Expansion of transfected cells transferred between the Cocoon™ andelectroporation Unit provided±10% variability in transfection efficiencywhen compared to the control culture 24 hours post transfection and onday of harvest, a final cell concentration≥80% of the control culture,±5% variability in Final Cell Viability when compared to the controlculture, ±10% variability when compared to the control culture in GFP+,CD3+, CD4+, and CD8+ expression, as determined via FACS, supernatant oftransferred transfected cells passed sterility testing and no mycoplasmadetected in pre- and post transfected culture samples.

Results

Cell Concentration in the Cocoon™ Cassette

The cells from two donors were concentrated by settling to a 10 mLvolume with 4.4×10⁸ and 4.2×10⁸ total viable cells. These two cellsuspensions were then diluted with 90 mL of supplemented electroporationSolution (NFS) and concentrated. Cell recovery post concentration was92% and 87%. Cell viability prior to transfection were 92% and 74% anddecreased by less than 5%. In both runs, 6% and 8% of the initialculture supernatant was detected in the final concentrated cellsuspension.

TABLE 3 Percent of detectable human serum A/B in the original culturesupernatant, post diluted and concentrated permeate, and final cellsuspension supernatant. Human Serum Human Serum Human Serum Human SerumHuman Serum Concentration of Concentration Concentration ConcentrationConcentration Initial Culture Pre Pre Post Post Sample ID (ng/mL)(ng/mL) (% of initial) (ng/mL) (% of initial) Donor 1 4.98E+06 2.19E+054% 2.84E+05 6.00% Donor 2 4.28E+06 3.48E+05 9% 3.30E+05 8.20%

There was no difference in CD4+:CD8+ profiles post concentrationcompared to the control culture that was not concentrated.

Results demonstrated recovery of fluids from the Cocoon™ satellite bagto the Cocoon™ output bag. Expansion of transfected cells weretransferred between the Cocoon™ and electroporation Unit. Successfulelectroporation is carried out in the electroporation Unit, resulting inthe transduced cells.

Automated, completely closed transfection using a closed loop betweenthe electroporation Unit and Cocoon™ systems, is provided herein.Methods can be used for concentration of cells in the Cocoon™ system.

Example 4—Hematopoietic Stem Cell Expansion

CD34+ focused on the expansion of cord blood. The specific applicationof this was the expansion of CD34+ from cord blood samples that containa low CD34+ number in order for single well-matched cords to be used foradult treatments. Therefore, the starting cell number and concentrationwas very low compared to some other protocols. It is expected that withlarger starting numbers and concentration, the cell expansion would belower.

CD34+ cells selected and expanded

Total nucleated cell (TNC) tracked over time

Starting cell concentrations were lower than many other protocols (0.1 Mcells/ml)

Cell expansion was found to vary based on collection protocol (FIG. 19).

Changes in cell phenotype are tracked during the culture period

25.3% of the TNC are CD34+ following 12 days of expansion (FIG. 20)

Differentiated cell phenotype is shown in FIG. 21. FIG. 22 demonstratesthat single colonies are capable of forming multi-lineagedifferentiation.

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It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein can be made without departing from thescope of any of the embodiments.

It is to be understood that while certain embodiments have beenillustrated and described herein, the claims are not to be limited tothe specific forms or arrangement of parts described and shown. In thespecification, there have been disclosed illustrative embodiments and,although specific terms are employed, they are used in a generic anddescriptive sense only and not for purposes of limitation. Modificationsand variations of the embodiments are possible in light of the aboveteachings. It is therefore to be understood that the embodiments may bepracticed otherwise than as specifically described.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

1. A cassette for use in an automated cell engineering system, thecassette comprising: a low temperature chamber for storage of a cellculture media; a high temperature chamber for carrying out activation,transduction and expansion of an immune cell culture; and a thermalbarrier, wherein the high temperature chamber is separated from the lowtemperature chamber by the thermal barrier, wherein the high temperaturechamber includes a cell culture chamber and one or more fluidicspathways connected to the cell culture chamber, and wherein the fluidicspathways provide recirculation, removal of waste and homogenous gasexchange and distribution of nutrients to the cell culture chamberwithout disturbing cells within the cell culture chamber.
 2. Thecassette of claim 1, wherein the low temperature chamber is maintainedfrom about 4° C. to about 8° C.
 3. The cassette of claim 1, wherein thehigh temperature chamber is maintained from about 35° C. to about 40° C.4. The cassette of claim 1, wherein the low temperature chamber canfurther include a bag or other holder for media, including about 1 L ormore of fluid.
 5. The cassette of claim 1, wherein the cassette furtherincludes one or more pumps for driving fluid through the cassette. 6.The cassette of claim 1, wherein the cassette further includes one ormore valves for controlling the flow through the fluidic pathways. 7.The cassette of claim 1, wherein the cassette further includes one ormore sampling ports and/or injection ports.
 8. The cassette of claim 1,wherein the cassette further includes one or more of a pH sensor, aglucose sensor, an oxygen sensor, a carbon dioxide sensor, a lactic acidsensor and/or an optical density sensor.
 9. A cassette for use in anautomated cell engineering system, the cassette comprising: a lowtemperature chamber for storage of a cell culture media; and a hightemperature chamber for carrying out activation, transduction andexpansion of an immune cell culture, wherein the high temperaturechamber includes: a flat and non-flexible cell culture chamber having achamber height of less than 5.0 cm, and one or more fluidics pathwaysconnected to the cell culture chamber, wherein the fluidics pathwaysprovide recirculation, removal of waste and homogenous gas exchange anddistribution of nutrients to the cell culture chamber without disturbingcells within the cell culture chamber.
 10. The cassette of claim 9,wherein the chamber height is about 0.5 cm to about 4.0 CM.
 11. Thecassette of claim 9, wherein the cell culture chamber has a volume ofabout 0.5 mL to about 300 mL.
 12. The cassette of claim 9, wherein thecell culture chamber further comprises at least one of: a distal portconfigured to allow for the removal of air bubbles from the cell culturechamber and/or as a recirculation port; a medial port configured tofunction as a recirculation inlet port; and a proximal port configuredto function as a drain port for cell removal.
 13. The cassette of claim9, wherein the cassette further includes one or more of a pH sensor, aglucose sensor, an oxygen sensor, a carbon dioxide sensor, a lactic acidsensor and/or an optical density sensor.
 14. A cassette for use in anautomated cell engineering system, the cassette comprising: a lowtemperature chamber for storage of a cell culture media; and a hightemperature chamber for carrying out activation, transduction andexpansion of an immune cell culture, wherein the high temperaturechamber includes: a cell culture chamber; and one or more fluidicspathways connected to the cell culture chamber, wherein the fluidicspathways provide recirculation, removal of waste and homogenous gasexchange and distribution of nutrients to the cell culture chamberwithout disturbing cells within the cell culture chamber, and whereinthe fluidics pathways comprise a silicon-based tubing component thatallows oxygenation through the tubing component.
 15. The cassette ofclaim 14, wherein the low temperature chamber can further include a bagor other holder for media, including about 1 L or more of fluid.
 16. Thecassette of claim 14, wherein the cassette further includes one or morepumps for driving fluid through the cassette.
 17. The cassette of claim14, wherein the cassette further includes one or more valves forcontrolling the flow through the fluidic pathways.
 18. The cassette ofclaim 14, wherein the cassette further includes one or more samplingports and/or injection ports.
 19. The cassette of claim 14, wherein thecassette further includes one or more of a pH sensor, a glucose sensor,an oxygen sensor, a carbon dioxide sensor, a lactic acid sensor and/oran optical density sensor.